U.S. patent application number 12/813297 was filed with the patent office on 2010-12-16 for immunological targeting of pathological tau proteins.
This patent application is currently assigned to NEW YORK UNIVERSITY. Invention is credited to Einar M. SIGURDSSON.
Application Number | 20100316564 12/813297 |
Document ID | / |
Family ID | 43306616 |
Filed Date | 2010-12-16 |
United States Patent
Application |
20100316564 |
Kind Code |
A1 |
SIGURDSSON; Einar M. |
December 16, 2010 |
IMMUNOLOGICAL TARGETING OF PATHOLOGICAL TAU PROTEINS
Abstract
The present invention relates to methods and compositions for
treating, preventing, and diagnosing Alzheimer's Disease or other
tauopathies in a subject by administering an immunogenic tau
peptide or an antibody recognizing the immunogenic tau epitope
under conditions effective to treat, prevent, or diagnose
Alzheimer's Disease or other tauopathies. Also disclosed are
methods of promoting clearance of aggregates from the brain of the
subject and of slowing progression of tau-pathology related
behavioral phenotype in a subject.
Inventors: |
SIGURDSSON; Einar M.;
(Scarsdale, NY) |
Correspondence
Address: |
NIXON PEABODY LLP - PATENT GROUP
1100 CLINTON SQUARE
ROCHESTER
NY
14604
US
|
Assignee: |
NEW YORK UNIVERSITY
New York
NY
|
Family ID: |
43306616 |
Appl. No.: |
12/813297 |
Filed: |
June 10, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61185895 |
Jun 10, 2009 |
|
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Current U.S.
Class: |
424/1.49 ;
424/139.1; 424/185.1; 424/9.4; 435/7.1; 436/501; 530/387.9 |
Current CPC
Class: |
A61P 25/16 20180101;
A61K 39/0005 20130101; G01N 2800/2821 20130101; A61P 37/04
20180101; A61K 39/3955 20130101; A61K 39/0007 20130101; A61K
2039/505 20130101; A61P 21/02 20180101; C07K 2317/20 20130101; A61P
25/00 20180101; A61P 25/14 20180101; C07K 16/18 20130101; G01N
33/6896 20130101; A61P 21/00 20180101; A61P 21/04 20180101; A61K
2039/507 20130101; A61P 43/00 20180101; A61P 25/28 20180101; C07K
2317/76 20130101 |
Class at
Publication: |
424/1.49 ;
424/185.1; 424/139.1; 530/387.9; 424/9.4; 435/7.1; 436/501 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 39/00 20060101 A61K039/00; C07K 16/18 20060101
C07K016/18; A61K 49/04 20060101 A61K049/04; A61K 51/10 20060101
A61K051/10; A61P 25/28 20060101 A61P025/28; A61P 25/00 20060101
A61P025/00; G01N 33/566 20060101 G01N033/566 |
Goverment Interests
[0002] The subject matter of this application was made with support
from the United States Government under the National Institutes of
Health, Grant No. AG032611. The U.S. Government has certain rights.
Claims
1. A method of treating or preventing Alzheimer's disease or other
tauopathy in a subject, said method comprising: administering to
the subject, one or more immunogenic tau peptides comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 2-75, or one or more antibodies recognizing an immunogenic tau
epitope comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2-75 and 101-103 under conditions
effective to treat or prevent Alzheimer's Disease or other
tauopathy in the subject.
2. The method according to claim 1 further comprising: selecting a
subject having or at risk of having Alzheimer's disease or other
tauopathy, wherein the one or more immunogenic tau peptides or one
or more antibodies recognizing an immunogenic tau epitope are
administered to the selected subject.
3. The method according to claim 1, wherein the one or more
immunogenic tau peptides are phosphorylated at one or more amino
acid residues and the one or more antibodies recognize a
phosphorylated immunogenic tau peptide.
4. The method according to claim 1, wherein an immunogenic carrier
is linked to the immunogenic tau peptide.
5. The method according to claim 1, wherein an adjuvant is
administered before, after, or concurrent with said administering
the one or more immunogenic tau peptides or antibodies.
6. The method according to claim 1, wherein one or more additional
immunogenic tau peptides are administered before, after, or
concurrent with said administering the one or more immunogenic tau
peptides.
7. The method according to claim 6, wherein the one or more
additional immunogenic tau peptides comprise an amino acid sequence
selected from the group consisting of SEQ ID NOs: 81-100.
8. The method according to claim 1, wherein a tauopathy is treated
or prevented, said tauopathy being selected from the group
consisting of frontotemporal dementia, parkinsonism linked to
chromosome 17 (FTDP-17), progressive supranuclear palsy,
corticobasal degeneration, Pick's disease, progressive subcortical
gliosis, tangle only dementia, diffuse neurofibrillary tangles with
calcification, argyrophilic grain dementia, amyotrophic lateral
sclerosis parkinsonism-dementia complex, dementia pugilistica, Down
syndrome, Gerstmann-Straussler-Scheinker disease,
Hallerworden-Spatz disease, inclusion body myositis,
Creutzfeld-Jakob disease, multiple system atropy, Niemann-Pick
disease type C, prion protein cerebral amyloid angiopathy, subacute
sclerosing panencephalitis, myotonic dystrophy, non-guanamian motor
neuron disease with neurofibrillary tangles, chronic traumatic
encephalopathy, and postencephalitic parkinsonism
9. A method of promoting clearance of tau aggregates from the brain
of a subject, said method comprising: administering to the subject,
one or more immunogenic tau peptides comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 2-75, or
one or more antibodies recognizing an immunogenic tau epitope
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2-75 and 101-103 under conditions
effective to promote clearance of the tau aggregates from the brain
of the subject.
10. The method according to claim 9 further comprising: selecting a
subject having tau aggregates in the brain, wherein the one or more
immunogenic tau peptides or one or more antibodies recognizing an
immunogenic tau epitope are administered to the selected
subject.
11. The method according to claim 9, wherein the one or more
immunogenic tau peptides are phosphorylated at one or more amino
acid residues and the one or more antibodies recognize a
phosphorylated immunogenic tau peptide.
12. The method according to claim 9, wherein an immunogenic carrier
is linked to the immunogenic tau peptide.
13. The method according to claim 9, wherein an adjuvant is
administered before, after, or concurrent with said administering
the one or more immunogenic tau peptides or antibodies.
14. The method according to claim 9, wherein one or more additional
immunogenic tau peptides are administered before, after, or
concurrent with said administering the one or more immunogenic tau
peptides.
15. The method according to claim 14, wherein the one or more
additional immunogenic tau peptides comprise an amino acid sequence
selected from the group consisting of SEQ ID NOs: 81-100.
16. The method according to claim 9, wherein the aggregates are
neurofibrillary tangles or their pathological tau precursors.
17. A method of slowing progression of a tau-pathology related
behavioral phenotype in a subject, said method comprising:
administering to the subject, one or more immunogenic tau peptides
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2-75, or one or more antibodies
recognizing an immunogenic tau epitope comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 2-75 and
101-103 under conditions effective to slow the progression of the
tau-pathology related behavioral phenotype in the subject.
18. The method according to claim 17 further comprising: selecting
a subject having a tau-pathology related behavioral phenotype,
wherein the one or more immunogenic tau peptides or one or more
antibodies recognizing an immunogenic tau epitope are administered
to the selected subject.
19. The method according to claim 17, wherein the one or more
immunogenic tau peptides are phosphorylated at one or more amino
acid residues and the one or more antibodies recognize a
phosphorylated immunogenic tau peptide.
20. The method according to claim 17, wherein an immunogenic
carrier is linked to the immunogenic tau peptide.
21. The method according to claim 17, wherein an adjuvant is
administered before, after, or concurrent with said administering
the one or more immunogenic tau peptides or antibodies.
22. The method according to claim 17, wherein one or more
additional immunogenic tau peptides are administered before, after,
or concurrent with said administering the one or more immunogenic
tau peptides.
23. The method according to claim 22, wherein the one or more
additional immunogenic tau peptides comprise an amino acid sequence
selected from the group consisting of SEQ ID NOs: 81-100.
24. An isolated tau peptide comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 2-75 and
101-103.
25. The isolated tau peptide according to claim 24, wherein the
peptide is phosphorylated at one or more amino acid residues.
26. The isolated tau peptide according to claim 24 further
comprising: an immunogenic carrier linked to the isolated
peptide.
27. A pharmaceutical composition comprising: the one or more
isolated peptides according to claim 24 and a pharmaceutical
carrier.
28. The pharmaceutical composition of claim 27 further comprising:
a pharmaceutically acceptable adjuvant.
29. The pharmaceutical composition of claim 27 further comprising:
one or more additional immunogenic tau peptides having an amino
acid sequence selected from the group consisting of SEQ ID NOs:
81-100.
30. An antibody or binding portion thereof having antigenic
specificity for an isolated tau peptide according to claim 24.
31. The antibody or binding portion thereof according to claim 30,
wherein the isolated peptide is phosphorylated.
32. The antibody or binding portion thereof according to claim 30,
wherein the antibody is a monoclonal antibody, a polyclonal
antibody, or an active binding portion thereof.
33. A combination immunotherapeutic comprising: the antibody
according to claim 30 and one or more antibodies or binding
portions thereof recognizing one or more different amyloidogenic
proteins or peptides.
34. The combination immunotherapeutic according to claim 33,
wherein the one or more amyloidogenic proteins or peptides is
selected from the group consisting of beta protein precursor, prion
and prion proteins, .alpha.-synuclein, amyloid-.beta., islet
amyloid polypeptide, apolipoprotein AI, apolipoprotein AII,
lyzozyme, cystatin C, gelsolin, atrial natriuretic factor,
calcitonin, keratoepithelin, lactoferrin, immunoglobulin light
chains, transthyretin, A amyloidosis, .beta.2-microglobulin,
immunoglobulin heavy chains, fibrinogen alpha chains, prolactin,
keratin, and medin.
35. A method of diagnosing Alzheimer's disease or other tauopathy
in a subject, said method comprising: detecting, in the subject,
the presence of a pathological tau protein conformer using a
diagnostic reagent, wherein the diagnostic reagent comprises an
antibody of claim 30, or an active binding fragment thereof, and
diagnosing Alzheimer's disease or other tauopathy in the subject
based on said detecting.
36. The method according to claim 35, wherein said detecting
comprising: obtaining a biological sample from the subject;
contacting the biological sample from the subject with the
diagnostic reagent under conditions effective for the diagnostic
reagent to bind to the pathological tau protein conformer in the
sample; and detecting binding of the diagnostic reagent to the
pathological tau protein conformer in the sample.
37. The method according to claim 35, wherein said detecting
comprises: administering the diagnostic reagent to the subject,
wherein the diagnostic reagent contains a detectable label and
detecting the labeled diagnostic reagent in the subject using an in
vivo imaging device.
38. A diagnostic kit comprising: the isolated antibody of claim 30
and a detectable label.
Description
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 61/185,895, filed Jun. 10, 2009, which
is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention is directed to immunological methods
and compositions for preventing, treating, and diagnosing
Alzheimer's disease and related tauopathies, and inhibiting the
accumulation of tau neurofibrillary tangles and/or their
pathological tau precursors in a subject.
BACKGROUND OF THE INVENTION
[0004] An emerging treatment for Alzheimer's disease (AD) is
immunotherapy to clear amyloid-.beta. (A.beta.). Another important
target in AD and frontotemporal dementia is the neurofibrillary
tangles and/or their pathological tau protein conformers, whose
presence correlates well with the degree of dementia (Terry R.,
"Neuropathological Changes in Alzheimer Disease," Prog Brain Res.
101:383-390 (1994); Goedert M., "Tau Protein and
Neurodegeneration," Semin Cell Dev Biol. 15:45-49 (2004)). The
objective of immunotherapy for tau pathology is that anti-tau
antibodies can clear tau aggregates that may affect neuronal
viability. Other components of the immune system may play a role as
well in the clearance. Tau is a soluble protein that promotes
tubulin assembly, microtubule stability, and cytoskeletal
integrity. Although tau pathology is likely to occur following
A.beta. aggregation based on Down syndrome studies, analyses of AD
brains and mouse models indicate that these pathologies are likely
to be synergistic (Sigurdsson et al., "Local and Distant
Histopathological Effects of Unilateral Amyloid-beta 25-35
Injections into the Amygdala of Young F344 Rats," Neurobiol Aging
17:893-901 (1996); Sigurdsson et al., "Bilateral Injections of
Amyloid-.beta. 25-35 into the Amygdala of Young Fischer Rats:
Behavioral, Neurochemical, and Time Dependent Histopathological
Effects," Neurobiol Aging 18:591-608 (1997); Lewis et al.,
"Enhanced Neurofibrillary Degeneration in Transgenic Mice
Expressing Mutant Tau and APP," Science 293(5534):1487-91 (2001);
Gotz et al., "Formation of Neurofibrillary Tangles in P301L Tau
Transgenic Mice Induced by A-beta 42 Fibrils," Science
293:1491-1495 (2001); Delacourte et al., "Nonoverlapping but
Synergetic Tau and APP Pathologies in Sporadic Alzheimer's
Disease," Neurology. 59:398-407 (2002); Oddo et al., "Abeta
Immunotherapy Leads to Clearance of Early, But Not Late,
Hyperphosphorylated Tau Aggregates via the Proteasome," Neuron
43:321-332 (2004); Ribe et al., "Accelerated Amyloid Deposition,
Neurofibrillary Degeneration and Neuronal Loss in Double Mutant
APP/Tau Transgenic Mice," Neurobiol Dis. (2005)). Hence, targeting
both pathologies may substantially increase treatment efficacy. To
date, no tau mutations have been observed in AD, however, in
frontotemporal dementia, mutations in the tau protein on chromosome
17 (FTDP-17) are a causative factor in the disease, which further
supports tau-based therapeutic approaches (Poorkaj et al., "Tau is
a Candidate Gene for Chromosome 17 Frontotemporal Dementia," Ann
Neurol. 43:815-825 (1998); Spillantini et al., "Frontotemporal
Dementia and Parkinsonism Linked to Chromosome 17: A New Group of
Tauopathies," Brain Pathol. 8:387-402 (1998)). Transgenic mice
expressing these mutations have modeled many aspects of the disease
and are valuable tools to study the pathogenesis of tau-pathology
related neurodegeneration and to assess potential therapies. One of
these models, the P301L mouse model (Lewis et al., "Neurofibrillary
Tangles, Amyotrophy and Progressive Motor Disturbance in Mice
Expressing Mutant (P301L) Tau Protein," Nat Genet. 25:402-405
(2000)), recapitulates many of the features of frontotemporal
dementia although the CNS distribution of the tau aggregates
results primarily in sensorimotor abnormalities which complicates
cognitive assessment. Homozygous lines of this mouse model have an
early onset of CNS pathology and associated functional impairments
which make them ideal for the initial assessment of the feasibility
of immunotherapy, targeting pathological tau conformers.
[0005] Other tau-related therapeutic approaches include: (1) drugs
that inhibit the kinases or activate the phosphatases that affect
the state of tau phosphorylation (Iqbal et al., "Inhibition of
Neurofibrillary Degeneration: A Promising Approach to Alzheimer's
Disease and Other Tauopathies," Curr Drug Targets 5:495-502 (2004);
Noble et al., Inhibition of Glycogen Synthase Kinase-3 by Lithium
Correlates with Reduced Tauopathy and Degeneration In Vivo," Proc
Natl Acad Sci USA 102:6990-6995 (2005)); (2) microtubule
stabilizing drugs (Michaelis et al., {beta}-Amyloid-Induced
Neurodegeneration and Protection by Structurally Diverse
Microtubule-Stabilizing Agents," J Pharmacol Exp Ther. 312:659-668
(2005); Zhang et al., "Microtubule-Binding Drugs Offset Tau
Sequestration by Stabilizing Microtubules and Reversing Fast Axonal
Transport Deficits in a Tauopathy Model," Proc Natl Acad Sci USA
102:227-231 (2005)); (3) compounds that interfere with tau
aggregation (Pickhardt et al., "Anthraquinones Inhibit Tau
Aggregation and Dissolve Alzheimer's Paired Helical Filaments In
Vitro and in Cells," J Biol. Chem. 280:3628-3635 (2005)); and (4)
drugs that promote heat shock protein mediated clearance of tau
(Dickey et al., "Development of a High Throughput Drug Screening
Assay for the Detection of Changes in Tau Levels--Proof of Concept
with HSP90 Inhibitors," Curr Alzheimer Res. 2:231-238 (2005)).
While all these approaches are certainly worth pursuing, target
specificity and toxicity are of a concern, which emphasizes the
importance of concurrently developing other types of tau-targeting
treatments, such as immunotherapy.
[0006] The present invention is directed to overcoming these and
other deficiencies in the art.
SUMMARY OF THE INVENTION
[0007] A first aspect of the present invention is directed to a
method of preventing or treating Alzheimer's disease or other
tauopathy in a subject. This method involves administering, to the
subject, any one or more immunogenic tau peptides having an amino
acid sequence selected from the group consisting of SEQ ID NOs:
2-75, or one or more antibodies recognizing an immunogenic tau
epitope comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2-75 and 101-103 under conditions
effective to treat or prevent Alzheimer's disease or other
tauopathy in the subject.
[0008] Another aspect of the present invention is directed to a
method of promoting clearance of tau aggregates from the brain of a
subject. This method involves administering, to the subject, any
one or more immunogenic tau peptides having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 2-75, or one or
more antibodies recognizing an immunogenic tau epitope comprising
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 2-75 and 101-103 under conditions effective to promote
clearance of the tau aggregates from the brain of the subject.
[0009] A third aspect of the present invention is directed to a
method of slowing progression of a tau-pathology related behavioral
phenotype in a subject. This method involves administering, to the
subject, any one or more immunogenic tau peptides having an amino
acid sequence selected from the group consisting of SEQ ID NOs:
2-75, or one or more antibodies recognizing an immunogenic tau
epitope comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2-75 and 101-103 under conditions
effective to slow the progression of the tau-pathology related
behavioral phenotype in the subject.
[0010] A fourth aspect of the present invention is directed to an
isolated tau peptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 2-75 and 101-103. The
immunogenic tau peptide is effective in preventing and treating
Alzheimer's disease or other tauopathy in a subject, promoting the
clearance of aggregates from the brain of a subject, and slowing
the progression of a tau-pathology related behavioral phenotype in
a subject.
[0011] Neurofibrillary tangles and their pathological tau protein
conformers are important targets for preventing and treating
Alzheimer's disease and other tau-related neurodegenerative
diseases. However, a strategy for targeting and clearing
neurofibrillary tangles and/or pathological tau conformers that has
high target specificity and minimal to no toxicity is lacking. The
immunogenic tau peptides and antibodies described herein were
designed to overcome this deficiency. Because the immunogenic tau
peptides of the present invention mimic narrow phospho-epitopes of
the pathological tau, and the tau antibodies recognize these same
narrow phospho-epitopes, enhanced specificity and safety are
achieved. This scenario also applies to the antibodies described
herein that are generated against the free N- or C-terminus of
pathological tau fragments. Accordingly, using the
immunotherapeutic approaches described herein, a robust immune
response against the pathological tau protein can be generated with
minimal risk of producing an adverse immune response towards the
normal tau protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIGS. 1A-1B depict the immune response in the JNPL3 P301L
tangle mouse model to tau immunogenic peptides of the present
invention. Mice of 2-3 months of age received the first two
immunizations two weeks apart and then monthly thereafter. To
assess antibody response, the mice were bled prior to the first
immunization, periodically thereafter one week after vaccine
administration, and when the mice were killed for tissue harvesting
at 8-9 months of age. The IgG and IgM antibody response shown in
FIGS. 1A and 1B was measured one week after the 6.sup.th
immunization (T3) and again at 8-9 months of age, which was at the
time of sacrifice (Tf=Tfinal). FIG. 1A shows a robust IgG and IgM
immune response in JNPL3 P301L mice immunized with
Tau210-216[P-Thr.sub.212-Ser.sub.214] (SEQ ID NO: 2) linked to
tetanus toxin helper T-cell epitope (TT947-967) via GPSL linker.
FIG. 1B shows that a strong antibody response is generated against
the tetanus toxin epitope as assessed by IgG and IgM binding to an
unrelated tau epitope Tau260-264[P-Ser.sub.262] linked via GPSL to
TT947-967. ELISA plates were coated with 0.5 .mu.g peptide per well
and plasma was diluted 1:200.
[0013] FIGS. 2A-2C show that JNPL3 P301L tangle mice immunized with
Tau260-264[P-Ser.sub.262] (SEQ ID NO: 3) (also referred to the T299
peptide) linked to tetanus toxin helper T-cell epitope (TT947-967)
via a GPSL linker generate a robust IgG response against the
immunogen. FIG. 2A shows the IgG antibody response in mice
following immunization with the Tau260-264[P-Ser.sub.262] peptide.
As above, the mice received the first two immunizations two weeks
apart and then monthly thereafter from 2-3 months of age until 8-9
months of age. FIG. 2B shows that a good portion of the antibody
response is generated against the tetanus toxin epitope as assessed
by IgG binding to an unrelated tau epitope
Tau210-216[P-Thr.sub.212-Ser.sub.214] linked via GPSL to TT947-967.
FIG. 2C shows that a good portion of the antibody response is
generated against the tau epitope as assessed by IgG binding to a
larger tau epitope Tau240-270[P-Ser.sub.262] that contains the
Tau260-264[P-Ser.sub.262] region. ELISA plates were coated with 0.5
.mu.g peptide per well and plasma was diluted 1:200. T0-Tfinal:
Bleed prior to vaccination (T0), one week after third -(T1), sixth
-(T2), seventh (T3) immunization, and at tissue harvesting
(Tf).
[0014] FIG. 3 shows the robust antibody (IgG) response generated in
JNPL3 P301L tangle model mice immunized in with
Tau229-237[P-Thr.sub.231-Ser.sub.235] (SEQ ID NO: 4) linked to
tetanus toxin helper T-cell epitope (TT947-967]. The mice were
immunized from 2-3 months of age, two weeks apart, then a month
later, and bled (T1) one week after the third immunization. ELISA
plates were coated with 0.5 .mu.g peptide per well and plasma was
diluted 1:200.
[0015] FIG. 4 shows the robust antibody (IgG) response generated in
JNPL3 P301L tangle model mice immunized with the
pseudophosphorylated immunogen, Tau379-408[Asp.sub.396, 404] (SEQ
ID NO: 57) in alum adjuvant. Importantly, these antibodies
recognize the phospho-epitope, Tau379-408[P-Ser.sub.396, 404], to a
similar degree. The mice were immunized from 2-3 months of age, two
weeks apart for the first two immunizations, and monthly
thereafter. The mice were bled (Tf=Tfinal) at the time of tissue
harvesting at 8-9 months of age. ELISA plates were coated with 0.5
.mu.g peptide per well and plasma was diluted 1:200.
[0016] FIG. 5A-5B show the reduction of pathological tau observed
in the brain stem (FIG. 5A) and dentate gyrus (FIG. 5B) of the
tangle mouse model following tau immunotherapy. Homozygous JNPL3
tau P301L mice were immunized with T299 (Tau260-264[P-Ser.sub.262]
(SEQ ID NO: 3)) linked to a tetanus toxin helper T-cell epitope
(TT947-967) via a GPSL linker sequence. Pathological tau in both
the brain stem and dentate gyrus were assessed by PHF1 antibody
immunostaining PHF1 is a monoclonal antibody recognizing tau that
is phosphorylated on serine amino acids 404 and 396 on the
C-terminal (Greenberg et al., "Hydrofluoric Acid-Treated Tau PHF
Proteins Display the Same Biochemical Properties as Normal Tau," J
Biol Chem 267:564-569 (1992), which is hereby incorporated by
reference in its entirety). A significant reduction of pathological
tau staining was observed in both the brain stem and dentate gyrus
of animals actively immunized with the T299 peptide compared to
control animals receiving adjuvant only.
[0017] FIG. 6 shows that immunization of htau/PS1 mice with the
phosphorylated Tau379-408[P-Ser.sub.396,404] (SEQ ID NO: 82)
reduces the amount of tau aggregates by 56% in the pyriform cortex.
Significant difference was observed between the immunized and
control groups (one-way ANOVA, p<0.01). Post hoc analysis also
showed that immunized htau/PS1 mice differed from their htau/PS1
controls (p<0.01). ** p<0.01.
[0018] FIGS. 7A-7B show that tau immunotherapy prevents functional
impairment in a tangle mouse model. Homozygous JNPL3 P301L mice
were immunized with the phosphorylated immunogenic Tau 299 peptide
(Tau260-264[P-Ser.sub.262] (SEQ ID NO:3)) linked to a tetanus toxin
helper T-cell epitope (TT947-967) via a GPSL linker sequence.
Control animals received adjuvant alone. Administration of the
Tau260-264[P-Ser.sub.262] peptide vaccine prevented functional
impairments assessed using the traverse beam at 8 months of age as
indicated by the fewer number of footslips recorded for the
immunized animals compared to the control animals (FIG. 7A).
Likewise, administration of the Tau260-264[P-Ser.sub.262] peptide
vaccine prevented functional impairments assessed by the rotarod
test, both at 5-6 months of age and at 8-9 months of age (FIG.
7B).
[0019] FIGS. 8A-8B show that immunization of htau/PS1 mice with the
phosphorylated Tau379-408[P-Ser.sub.396,404] (SEQ ID NO:82)
improves performance in the radial arm maze (FIG. 8A) and the
object recognition test (FIG. 8B). A significant difference was
observed between the immunized and control groups in the radial arm
maze (two-way ANOVA repeated measures, p<0.0001) as shown in
FIG. 8A. Neuman-Keuls post-hoc test revealed that the immunized
htau/PS1 mice performed better (i.e., committed less errors) than
the control htau/PS1 mice on all the days (p<0.01-0.001). A
significant difference was also observed between the groups in the
object recognition test (one-way ANOVA, p=0.005) (FIG. 8B).
Neuman-Keuls post-hoc test revealed that the immunized htau/PS1
mice had better short-term memory than identical control mice
(p<0.01). It has been well established that cognitively normal
mice spend about 70% of their time with the new object compared to
the old object. ** p<0.01.
[0020] FIGS. 9A-9C show that immunization of htau/PS1 mice with the
phosphorylated Tau379-408[P-Ser.sub.396,404] (SEQ ID NO:82)
improves performance in the closed field symmetrical maze.
Significant differences were observed between the immunized and
control groups with respect to the number of errors committed in
each of mazes 9A-9C (one-way ANOVA, Maze A: p<0.001, Maze B:
p<0.0001, Maze C: p<0.01). Post-hoc analysis revealed that
the treated htau/PS1 group performed better than their identical
control mice (htau/PS1 controls) (Maze A: p<0.01, Mazes B, C:
p<0.001). Post-hoc analysis also revealed significant
differences between some of the other groups depending on the maze
but those differences are less relevant and are therefore not
detailed here. The three mazes were of increasing complexity as
indicated by the number of errors (note that the Y axis scale
differs). ** p<0.01, *** p<0.001.
[0021] FIGS. 10A-10F are graphs depicting the levels of soluble and
insoluble tau (total tau and pathological tau) detected by western
blot analysis in htau/PS1 mice immunized with phosphorylated
Tau379-408[P-Ser.sub.396,404] (SEQ ID NO:82) and corresponding
controls. Tau immunotherapy reduces pathological tau compared to
total tau by 35-43% (FIGS. 10C and 10D). The immunotherapy did not
affect total tau levels as assessed with B19 antibody (FIGS. 10A
and 10B) which is important for the safety of this approach.
Compared to htau/PS1 controls, PHF1 soluble tau was significantly
reduced (p<0.001) and the soluble tau ratio (PHF1/total tau) was
reduced by 35% (p<0.05) (FIG. 10E). A strong trend for reduction
in PHF1 insoluble tau was observed as well (p=0.06), and the
insoluble tau ratio (PHF1/total tau) was reduced by 43% (p=0.08)
(FIG. 10F). * p<0.05, *** p<0.001
[0022] FIGS. 11A-11C demonstrate that passive immunotherapy
targeting the phosphorylated tau 396 and 404 epitopes prevents
functional decline and reduces tau pathology in P301L tangle mice.
FIG. 11A is a graph showing a significant difference in the number
of footslips taken on the traverse beam by IgG injected control and
PHF1 immunized P301L mice, with control animals having more
footslips when crossing the beam (trials combined, p=0.03). FIG.
11B is a graph showing the percentage of tau immunostaining in the
dentate gyrus of immunized and control P301L mice. PHF1 immunized
P301L mice had 58% less PHF1 stained tau pathology in the dentate
gyrus than controls (p=0.02). As shown in FIG. 11C, the amount of
PHF-1 antibodies (.mu.g/.mu.L) in plasma decreased four-fold in two
weeks. No detectable antibodies were observed in controls, whereas
the levels in immunized animals decreased over time. These are the
average values for the immunized mice. T0: prior to first
immunization, T1: 24 h after the 12th injection, T2: 7 days after
the 13th and last injection, T3: 14 days after last injection. The
ELISA plates were coated with Tau379-08[P-Ser.sub.396,404]
[0023] FIGS. 12A-12B are graphs showing the inverse correlation
between plasma levels of PHF 1 antibodies and tau pathology.
Significant correlation was observed in the brain stem (FIG. 12A;
p<0.01), and a strong trend for correlation in the motor cortex
(FIG. 12B; p=0.06).
[0024] FIGS. 13A-13B are graphs depicting the generation of
monoclonal antibodies against the immunogenic tau peptide
comprising amino acids 386-408 (SEQ ID NO:13) containing
phosphorylated serine epitopes at amino acid positions 396 and 404.
As shown in FIG. 13A, a very strong titer was generated against the
tau portion of the immunogen Tau-386-408[P-Ser.sub.396, 404] (red)
as detected by serial dilutions of plasma. The plasma antibodies
preferably recognized the phospho-Ser404 epitope (blue) and the
non-phospho epitope (white). The phospho-Ser396 epitope (green) was
recognized to a lesser degree. Numerous strongly positive clones
were detected (>50). Of those, 8 phospho-specific clones were
selected for a first subcloning (FIG. 13B). All appeared stable and
three were selected for second subcloning (all IgG1). Of the clones
that did not specifically recognize a phosphorylated-epitope, six
were selected for first subcloning. All appeared stable and three
were selected for second subcloning (IgG1, IgG2a and IgM).
[0025] FIGS. 14A-14B are graphs showing epitope binding of stable
phospho-specific (FIG. 14A) and non-phospho-specific (FIG. 14B)
Tau-386-408[P-Ser.sub.396, 404] antibody clones after third
subcloning by ELISA. Of the phospho-specific monoclonal antibodies
selected for further subcloning, four out of six retained their
specificity for the phospho-Ser.sub.404 epitope (see clones 1F12C2,
1F12G6, 4E6E3, and 4E6G7 in FIG. 14A). Two clones are less
phospho-specific (8B2D1) or non-specific (8B2D4) (FIG. 14A). Of the
non-phospho-specific monoclonal antibodies, 6B2E9 and 6B2G12, in
particular, retained their non-specificity after further subcloning
(FIG. 14B). Data presented was obtained at 1:810 dilution of
culture supernatant.
[0026] FIGS. 15A-15B are western blots showing reactivity of the
four Tau-386-408[P-Ser.sub.396, 404] phospho-specific (FIG. 15A)
and non-phospho-specific (FIG. 15B) monoclonal antibody clones with
brain homogenates from the JNPL3 P301L mouse and wildtype (Wt)
mouse. Of the four phospho-specific clones, 4E6G7 shows the
strongest reactivity, which is consistent with the ELISA results of
FIG. 14A. In contrast with the PHF-1 antibody that also recognizes
the tau P-Ser.sub.396, 404 epitope, all clones react better with
the JNPL3 P301L brain homogenate than the Wt homogenate. The
non-phospho-specific clones reacted faster, as expected, as most of
tau is non-phosphorylated.
[0027] FIGS. 16A-16B illustrate the generation of monoclonal
antibodies against the immunogenic tau peptide comprising amino
acids 260-271 (SEQ ID NO:12) and containing phosphorylated serine
262 epitope. As shown in FIG. 16A, a strong titer was generated
against the immunogen Tau260-271[P-Ser.sub.262] (purple), but
plasma antibodies recognized the non-phospho peptide Tau260-271 as
well (No-P; white). Eight stable phospho-specific clones were
selected for further analysis (FIG. 16B).
[0028] FIG. 17 is a western blot showing the reactivity of the
three phospho-specific Tau260-271[P-Ser.sub.262] monoclonal
antibody clones. The 2C11 antibody clone recognizes a higher
molecular weight band than the other phospho-specific clones and it
does not distinguish between wildtype and P301L tissue. 5F7D10 and
5F7E9 are representatives of the other clones. Tau-5 recognizes
total tau and binds to an epitope around amino acids 216-227 of
tau. CP27 recognizes human but not mouse tau.
[0029] FIGS. 18A-18E are immunohistochemical photomicrographs
showing the detection of tau pathology using the 5F7D10 antibody
clone in P301L tangle mouse brain sections. The 5F7D10 monoclonal
antibody shows strong histological staining in the P301L brain
section (FIG. 18A) compared to the wildtype (FIG. 18B). The PHF1
antibody picked up tau pathology in the same tangle mouse (FIG.
18C) although the pattern was different than with the 5F7D10
antibody, which is not surprising as they recognize different tau
epitopes. FIG. 18D is a magnified image of the boxed region in FIG.
18A depicting neurons with aggregated tau. FIG. 18E is a higher
magnified image of tangle-like pathology detected with 5F7D10 in a
different JNPL3 P301L mouse.
DETAILED DESCRIPTION OF THE INVENTION
[0030] A first aspect of the present invention is directed to a
method of preventing or treating Alzheimer's disease or other
tauopathy in a subject. This method involves administering, to the
subject, any one or more immunogenic tau peptides having an amino
acid sequence selected from the group consisting of SEQ ID NOs:
2-75, or one or more antibodies recognizing an immunogenic tau
epitope comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 2-75 and 101-103 under conditions
effective to treat or prevent Alzheimer's disease or other
tauopathy in the subject.
[0031] As used herein a "tauopathy" encompasses any
neurodegenerative disease that involves the pathological
aggregation of the microtubule protein tau within the brain.
Accordingly, in addition to both familial and sporadic Alzheimer's
disease, other tauopathies that can be treated using the methods of
the present invention include, without limitation, frontotemporal
dementia, parkinsonism linked to chromosome 17 (FTDP-17),
progressive supranuclear palsy, corticobasal degeneration, Pick's
disease, progressive subcortical gliosis, tangle only dementia,
diffuse neurofibrillary tangles with calcification, argyrophilic
grain dementia, amyotrophic lateral sclerosis parkinsonism-dementia
complex, dementia pugilistica, Down syndrome,
Gerstmann-Straussler-Scheinker disease, Hallerworden-Spatz disease,
inclusion body myositis, Creutzfeld-Jakob disease, multiple system
atropy, Niemann-Pick disease type C, prion protein cerebral amyloid
angiopathy, subacute sclerosing panencephalitis, myotonic
dystrophy, non-guanamian motor neuron disease with neurofibrillary
tangles, postencephalitic parkinsonism, and chronic traumatic
encephalopathy.
[0032] Another aspect of the present invention is directed to a
method of promoting clearance of tau aggregates from the brain of a
subject. This method involves administering, to the subject, any
one or more immunogenic tau peptides having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 2-75, or one or
more antibodies recognizing an immunogenic tau epitope comprising
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 2-75 and 101-103 under conditions effective to promote
clearance of tau aggregates from the brain of the subject.
[0033] The clearance of tau aggregates includes clearance of
neurofibrillary tangles and/or the pathological tau precursors to
neurofibrillary tangles. Neurofibrillary tangles are often
associated with neurodegenerative diseases including, for example,
sporadic and familial Alzheimer's disease, amyotrophic lateral
sclerosis, argyrophilic grain dementia, dementia pugilistica,
chronic traumatic encephalopathy, diffuse neurofibrillary tangles
with calcification, Down syndrome, Gerstmann-Straussler-Scheinker
disease, Hallervorden-Spatz disease, hereditary frontotemporal
dementia, parkinsonism linked to chromosome 17 (FTDP-17), inclusion
body myositis, Creutsfeld-Jakob disease, multiple system atrophy,
Niemann-Pick disease type C, Pick's disease, prion protein cerebral
amyloid angiopathy, sporadic corticobasal degeneration, progressive
supranuclear palsy, subacute sclerosing panencephalitis, myotonic
dystrophy, motor neuron disease with neurofibrillary tangles,
tangle only dementia, and progressive subcortical gliosis.
[0034] Another aspect of the present invention is directed to a
method of slowing the progression of a tau-pathology related
behavioral phenotype in a subject. This method involves
administering, to the subject, any one or more immunogenic tau
peptides comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 2-75, or one or more antibodies
recognizing an immunogenic tau epitope comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 2-75 and
101-103, under conditions effective to slow the tau-pathology
related behavioral phenotype in the subject.
[0035] As used herein, a tau-pathology related behavioral phenotype
includes, without limitation, cognitive impairments, early
personality change and disinhibition, apathy, abulia, mutism,
apraxia, perseveration, stereotyped movements/behaviors,
hyperorality, disorganization, inability to plan or organize
sequential tasks, selfishness/callousness, antisocial traits, a
lack of empathy, halting, agrammatic speech with frequent
paraphasic errors but relatively preserved comprehension, impaired
comprehension and word-finding deficits, slowly progressive gait
instability, retropulsions, freezing, frequent falls, non-levodopa
responsive axial rigidity, supranuclear gaze palsy, square wave
jerks, slow vertical saccades, pseudobulbar palsy, limb apraxia,
dystonia, cortical sensory loss, and tremor.
[0036] In accordance with the methods of the present invention, in
one embodiment, an immunogenic tau peptide or a combination of
immunogenic tau peptides are administered to a subject in need.
Suitable immunogenic tau peptide fragments of the tau protein
contain one or more antigenic epitopes that mimic the pathological
form of the tau protein. Exemplary immunogenic tau epitopes are
phosphorylated at one or more amino acids that are phosphorylated
in the pathological form of tau, but not phosphorylated in the
normal or non-pathological form of tau.
[0037] In a preferred embodiment of the present invention,
administration of an immunogenic tau peptide induces an active
immune response in the subject to the immunogenic tau peptide and
to the pathological form of tau, thereby facilitating the clearance
of related tau aggregates, slowing the progression of tau-pathology
related behavior and treating the underlying tauopathy. In
accordance with this aspect of the present invention, an immune
response involves the development of a beneficial humoral (antibody
mediated) and/or a cellular (mediated by antigen-specific T cells
or their secretion products) response directed against the
immunogenic tau peptide.
[0038] The presence of a humoral immunological response can be
determined and monitored by testing a biological sample (e.g.,
blood, plasma, serum, urine, saliva feces, CSF or lymph fluid) from
the subject for the presence of antibodies directed to the
immunogenic tau peptide. Methods for detecting antibodies in a
biological sample are well known in the art, e.g., ELISA, Dot
blots, SDS-PAGE gels or ELISPOT. The presence of a cell-mediated
immunological response can be determined by proliferation assays
(CD4.sup.+ T cells) or CTL (cytotoxic T lymphocyte) assays which
are readily known in the art.
[0039] Isolated immunogenic tau peptides of the present invention
include any one of the amino acid sequences of SEQ ID NOs: 2-30
shown in Table 1 below. Amino acid residues of each sequence which
are phosphorylated are shown in bold and marked with asterisks. The
names of the peptides in Table 1 correspond to the amino acid
position of these peptides within the longest isoform of the human
tau protein having the amino acid sequence of SEQ ID NO:1 as shown
below.
TABLE-US-00001 Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp
His Ala Gly 1 5 10 15 Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly
Gly Tyr Thr Met His 20 25 30 Gln Asp Gln Glu Gly Asp Thr Asp Ala
Gly Leu Lys Glu Ser Pro Leu 35 40 45 Gln Thr Pro Thr Glu Asp Gly
Ser Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60 Asp Ala Lys Ser Thr
Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val 65 70 75 80 Asp Glu Gly
Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu 85 90 95 Ile
Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro 100 105
110 Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val
115 120 125 Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala
Lys Gly 130 135 140 Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly
Ala Ala Pro Pro 145 150 155 160 Gly Gln Lys Gly Gln Ala Asn Ala Thr
Arg Ile Pro Ala Lys Thr Pro 165 170 175 Pro Ala Pro Lys Thr Pro Pro
Ser Ser Gly Glu Pro Pro Lys Ser Gly 180 185 190 Asp Arg Ser Gly Tyr
Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser 195 200 205 Arg Ser Arg
Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys 210 215 220 Lys
Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys 225 230
235 240 Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn
Val 245 250 255 Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln
Pro Gly Gly 260 265 270 Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp
Leu Ser Asn Val Gln 275 280 285 Ser Lys Cys Gly Ser Lys Asp Asn Ile
Lys His Val Pro Gly Gly Gly 290 295 300 Ser Val Gln Ile Val Tyr Lys
Pro Val Asp Leu Ser Lys Val Thr Ser 305 310 315 320 Lys Cys Gly Ser
Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln 325 330 335 Val Glu
Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser 340 345 350
Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn 355
360 365 Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys
Ala 370 375 380 Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro
Val Val Ser 385 390 395 400 Gly Asp Thr Ser Pro Arg His Leu Ser Asn
Val Ser Ser Thr Gly Ser 405 410 415 Ile Asp Met Val Asp Ser Pro Gln
Leu Ala Thr Leu Ala Asp Glu Val 420 425 430 Ser Ala Ser Leu Ala Lys
Gln Gly Leu 435 440
TABLE-US-00002 TABLE 1 Immunogenic Tau Peptides SEQ ID NO: NAME
SEQUENCE SEQ ID NO: 2 Tau210-216 [P-Thr.sub.212- SRT*PS*LP
Ser.sub.214] SEQ ID NO: 3 Tau260-264 [P-Ser.sub.262] IGS*TE SEQ ID
NO: 4 Tau229-237 [P-Thr.sub.231- VRT*PPKS*PS Ser.sub.235] SEQ ID
NO: 5 Tau394-406 [P-Ser.sub.396,404] YKS*PVVSGDTS*PR SEQ ID NO: 6
Tau192-221 [P-Thr.sub.212 Ser.sub.214]
GDRSGYSSPGSPGTPGSRSRT*PS*LPTPPTR SEQ ID NO: 7 Tau192-221
[P-Ser.sub.199, 202, 214, GDRSGYSS*PGS*PGT*PGSRSRT*PS*LPTPPTR
Thr.sub.205, 212] SEQ ID NO: 8 Tau192-221 [P-Ser.sub.199,214
GDRSGYSS*PGSPGTPGSRSRT*PS*LPT*PPTR Thr.sub.212, 217] SEQ ID NO: 9
Tau192-221 [P-Ser.sub.202Thr.sub.205]
GDRSGYSSPGS*PGT*PGSRSRTPSLPTPPTR SEQ ID NO: 10 Tau200-229
[P-Thr.sub.212- PGSPGTPGSRSRT*PS*LPTPPTREPKKVAVV Ser.sub.214] SEQ
ID NO: 11 Tau322-358[P-Ser.sub.324,356]
CGS*LGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGS*LD SEQ ID NO: 12 Tau260-271
[P-Ser.sub.262] IGS*TENLKHQPG SEQ ID NO: 13 Tau386-408
[P-Ser.sub.396, TDHGAEIVYKS*PVVSGDTS*PRHL Ser.sub.404] SEQ ID NO:
14 Tau48-71 [P-Thr.sub.50,69] LQT*PTEDGSEEPGSETSDAKST*PT SEQ ID NO:
15 Tau111-115 [P-Ser.sub.113] TPS*LE SEQ ID NO: 16
Tau151-155[P-Thr.sub.153] IAT*PR SEQ ID NO: 17
Tau173-177[P-Thr.sub.175] AKT*PP SEQ ID NO: 18
Tau203-219[P-Thr.sub.205,212,217- PGT*PGS*RS*RT*PS*LPT*PP
Ser.sub.208,210,214 SEQ ID NO: 19 Tau233-237[P-Thr.sub.235] PKS*PS
SEQ ID NO: 20 Tau256-264[P-Ser.sub.258,262] VKS*KIGS*TE SEQ ID NO:
21 Tau287-291[P-Ser.sub.289] VQS*KC SEQ ID NO: 22
Tau354-358[P-Ser.sub.356] IGS*LD SEQ ID NO: 23
Tau398-416[P-S.sub.400,409,412,413- VVS*GDVT*SPRHLS*NVS*S*T*GS
Thr.sub.403,414] SEQ ID NO: 24 Tau420-437[P-Ser.sub.422,433,435-
VDS*PQLAVT*LADEVS*AS*LA Thr.sub.427] SEQ ID NO: 25
Tau200-204[P-Ser.sub.202] PGS*P SEQ ID NO: 26
Tau203-207[P-Thr.sub.205] PGT*PG SEQ ID NO: 27
Tau197-207[P-Ser.sub.199,202- YSS*PGS*PGT*PG Thr205] SEQ ID NO: 28
Tau206-216 [P-Thr.sub.212- PGSRSRT*PS*LP Ser.sub.214] SEQ ID NO: 29
Tau229-239 [P-Thr.sub.231- VRT*PPKS*PSSA Ser.sub.235] SEQ ID NO: 30
Tau179-188 [P-Thr.sub.181- PKT*PPS*S*GEP Ser.sub.184,185]
[0040] Variants and analogs of the above immunogenic peptides that
induce and/or crossreact with antibodies to the preferred epitopes
of tau protein can also be used. Analogs, including allelic,
species, and induced variants, typically differ from naturally
occurring peptides at one, two, or a few positions, often by virtue
of conservative substitutions. Analogs typically exhibit at least
80 or 90% sequence identity with natural peptides. Some analogs
also include unnatural amino acids or modifications of N- or
C-terminal amino acids at one, two, or a few positions.
[0041] In one embodiment of the present invention, variant
immunogenic tau peptides are pseudo-phosphorylated peptides. The
pseudo-phosphorylated peptides are generated by substituting one or
more of the phosphorylated serine, threonine, and tyrosine residues
of the tau peptides with acidic amino acid residues such as
glutamic acid and aspartic acid (Huang et al., "Constitutive
Activation of Mekl by Mutation of Serine Phosphorylation Sites,"
Proc. Natl. Acad. Sci. USA 91(19):8960-3 (1994), which is hereby
incorporated by reference in its entirety). Exemplary isolated
immunogenic pseudo-phosphorylated tau peptides of the present
invention are shown in Table 2 below. The position of the amino
acid residue substitutions is indicated in each sequence of Table 2
with an "X", where X is an glutamic acid or aspartic acid residue
substitution.
TABLE-US-00003 TABLE 2 Immunogenic Pseudo-Phosphorylated Tau
Peptides SEQ ID NO: NAME SEQUENCE SEQ ID NO: 31 Tau210-216 [T212X,
SRXPXLP S214X] SEQ ID NO: 32 Tau260-264 [S262X] IGXTE SEQ ID NO: 33
Tau229-237 [T231X, VRXPPKXPS S235X] SEQ ID NO: 34 Tau394-406
[S396X, S202X] YKXPVVSGDTXPR SEQ ID NO: 35 Tau192-221 [T212X,
S214X] GDRSGYSSPGSPGTPGSRSRXPXLPTPPTR SEQ ID NO: 36 Tau192-221
[S199X, S202X, GDRSGYSXPGXPGXPGSRSRXPXLPTPPTR S214X, T205X, T212X]
SEQ ID NO: 37 Tau192-221 [S199X, S214X,
GDRSGYSXPGSPGTPGSRSRXPXLPXPPTR T212X, T217X] SEQ ID NO: 38
Tau192-221 [S202X, GDRSGYSSPGXPGXPGSRSRTPSLPTPPTR T205X] SEQ ID NO:
39 Tau200-229 [T212X, S214X] PGSPGTPGSRSRXPXLPTPPTREPKKVAVV SEQ ID
NO: 40 Tau322-358[S324X, S356X]
CGXLGNIHHKPGGGQVEVKSEKLDFKDRVQSKIGXLD SEQ ID NO: 41
Tau260-271[S262X] IGXTENLKHQPG SEQ ID NO: 42 Tau386-408 [S396X,
S404X] TDHGAEIVYKXPVVSGDTXPRHL SEQ ID NO: 43 Tau48-71 [T50X, T69X]
LQXPTEDGSEEPGSETSDAKSXPT SEQ ID NO: 44 Tau111-115 [S113X] TPXLE SEQ
ID NO: 45 Tau151-155[T153X] IAXPR SEQ ID NO: 46 Tau173-177[T175X]
AKXPP SEQ ID NO: 47 Tau203-219[T205X, T212X, PGXPGXRXRXPXLPXPP
T217X, S208X, S210X, S214X] SEQ ID NO: 48 Tau233-237[T235X] PKXPS
SEQ ID NO: 49 Tau256-264[S258X, S262X] VKXKIGXTE SEQ ID NO: 50
Tau287-291[S289X] VQXKC SEQ ID NO: 51 Tau354-358[S356X] IGXLD SEQ
ID NO: 52 Tau398-416[S400X, S409X, VVXGDXSPRHLXNVXXXGS S412X,
S413X, T403X, S414X] SEQ ID NO: 53 Tau420-437[S422X, S433X,
VDXPQLAXLADEVXAXLA S435X, T427X] SEQ ID NO: 54 Tau200-204[S202X]
PGXP SEQ ID NO: 55 Tau203-207[T205X] PGXPG SEQ ID NO: 56 Tau
133-162 [T149X, DGTGSDDKKAKGADGKXKIAXTPRGAAPPGQ T153X] SEQ ID NO:
57 Tau 379-408 [S396X, RENAKAKTDHGAEIVYKXPVVSGDTXPRHL S404X] SEQ ID
NO: 58 Tau 192-221 [S199X, GDRSGYSXPGXPGXPGSRSRXPXLPTPPTR S202X,
S214X, T205X, T212X] SEQ ID NO: 59 Tau221-250 [T231X,
REPKKVAVVRXPPKXPSSAKSRLQTAPVPM S235X] SEQ ID NO: 60
Tau184-213[S184X, S191X, XSGEPPKXGDRSQXXXPGXPGXPGXRXRX Y197X,
S198X, S199X, S202X, T205X, S208X, S210X, T212X] SEQ ID NO: 61
Tau1-30 [Y18X, Y29X] MAEPRQEFEVMEDHAGTXGLGDRKDQGGXT SEQ ID NO: 62
Tau30-60 [T39X, S46X, TMHQDQEGDXDAGLKEXPLQXPXEDGXEEPG T50X, T52X,
S56X] SEQ ID NO: 63 Tau60-90 [S68X, T69X,
GSETSDAKXXPXAEDVTAPLVDEGAPGKQAA T71X] SEQ ID NO: 64 Tau90-120
[T95X, T101X, AAQPHXEIPEGXXAEEAGIGDTPXLEDEAAG T102X, T113X] SEQ ID
NO: 65 Tau120-150 [T123X, S131X, GHVXQARMVSKXKDGTGSDDKKAKGADGKXK
T149X] SEQ ID NO: 66 Tau150-180 [T175X]
KIATPRGAAPPGQKGQANATRIPAKXPPAPK SEQ ID NO: 67 Tau180-210 [T181X,
S184X, KXPPXXGEPPKSGDRSGXXXPGXPGXPGXRS S185X, Y197X, S198X, S199X,
S202X, T205X, S208X] SEQ ID NO: 68 Tau210-240 [T212X, S214X,
SRXPXLPXPPTREPKKVAVVRXPPKXPXXAK T217X, T231X, S235X, S237X, S238X]
SEQ ID NO: 69 Tau240-270 [S262X] KSRLQTAPVPMPDLKNVKSKIGXTENLKHQP
SEQ ID NO: 70 Tau270-300 [S293] PGGGKVQIINKKLDLSNVQSKCGXKDNIKHV SEQ
ID NO: 71 Tau300-330 [Y310, S324X] VPGGGSVQIVXKPVDLSKVTSKCGXLGNIHH
SEQ ID NO: 72 Tau330-360 [S356X] HKPGGGQVEVKSEKLDFKDRVQSKIGXLDNI
SEQ ID NO: 73 Tau360-390 [T361X, T373X,
IXHVPGGGNKKIEXHKLTFRENAKAKXDHGA T386X] SEQ ID NO: 74 Tau390-420
[Y394X, AEIVXKXPVVXGDXXPRHLXNVXXTGSIDMV S396X, S400X, T403X, T404X,
S409X, S412X, S413X] SEQ ID NO: 75 Tau411-441 [S412X, S413X,
VXXTGSIDMVDXPQLATLADEVSASLAKQGL S422X]
[0042] Each tau peptide of the present invention, i.e., SEQ ID NOs:
2-75 and 87-88 (Table 3 below) is preferably acetylated on the
N-terminus and amidated on the C-terminus to more closely resemble
the same internal amino acids of the full length tau protein. The
tau peptides of the present invention can also contain one or more
D-amino acid residues to enhance the stability of the peptide.
These D-amino acids can be in the same order as the L-form of the
peptide or assembled in a reverse order from the L-form sequence to
maintain the overall topology of the native sequence (Ben-Yedidia
et al., "A Retro-Inverso Peptide Analogue of Influenza Virus
Hemagglutinin B-cell Epitope 91-108 Induces a Strong Mucosal and
Systemic Immune Response and Confers Protection in Mice after
Intranasal Immunization," Mol Immunol. 39:323 (2002); Guichard, et
al., "Antigenic Mimicry of Natural L-peptides with
Retro-Inverso-Peptidomimetics," PNAS 91:9765-9769 (1994);
Benkirane, et al., "Antigenicity and Immunogenicity of Modified
Synthetic Peptides Containing D-Amino Acid Residues," J. Bio. Chem.
268(35):26279-26285 (1993), which are hereby incorporated by
reference in their entirety).
[0043] Each of the above peptide sequences may be linked to an
immunogenic carrier molecule to enhance its immunogenicity.
Suitable immunogenic carrier molecules include, but are not limited
to, helper T-cell epitopes, such as tetanus toxoid (e.g., the P2
and P30 epitopes), Hepatitis B surface antigen, cholera toxin B,
toxoid, diphtheria toxoid, measles virus F protein, Chlamydia
trachomatis major outer membrane protein, Plasmodium falciparum
circumsporozite T, P. falciparum CS antigen, Schistosoma mansoni
triose phosphate isomerase, Bordetella pertussis, Clostridium
tetani, Pertusaria trachythallina, Escherichia coli TraT, and
Influenza virus hemagluttinin (HA) (see U.S. Pat. No. 6,906,169 to
Wang; U.S. Patent Application Publication No. 20030068325 to Wang,
and WO/2002/096350 to Wang, which are hereby incorporated by
reference in their entirety). In a preferred embodiment of the
present invention, the T-helper cell epitope is the tetanus toxin
947-967 (P30) epitope having an amino acid sequence of
FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 76). In another embodiment, the
T-helper cell epitope is the tetanus toxin 830-843 (P2) epitope
having an amino acid sequence of QYIKANSKFIGIT (SEQ ID NO: 77).
[0044] The immunogenic tau peptides of the present invention can be
linked to the immunogenic carrier molecule using a short amino acid
linker sequence. In a preferred embodiment of the present
invention, a GPSL (SEQ ID NO: 78) linker sequence is used to link
the immunogenic tau peptide to the immunogenic carrier molecule.
Other suitable linker sequences include glycine-rich (e.g.
G.sub.3-5) or serine-rich (e.g., GSG, GSGS (SEQ ID NO: 79), GSGSG
(SEQ ID NO: 80), GS.sub.NG) linker sequences or flexible
immunoglobulin linkers as disclosed in U.S. Pat. No. 5,516,637 to
Huang et al, which is hereby incorporated by reference in its
entirety.
[0045] Alternatively, the immunogenic tau peptides of the present
invention can be linked to the immunogenic carrier molecule using
chemical crosslinking Techniques for linking a peptide immunogen to
an immunogenic carrier molecule include the formation of disulfide
linkages using N-succinimidyl-3-(2-pyridyl-thio) propionate (SPDP)
and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(SMCC) (if the peptide lacks a sulfhydryl group, this can be
provided by addition of a cysteine residue). These reagents create
a disulfide linkage between themselves and peptide cysteine
residues on one protein, and an amide linkage through the
epsilon-amino on a lysine, or other free amino group in other amino
acids. A variety of such disulfide/amide-forming agents are
described by Jansen et al., "Immunotoxins: Hybrid Molecules
Combining High Specificity and Potent Cytotoxicity," Immun Rev
62:185-216 (1982), which is incorporated by reference in its
entirety. Other bifunctional coupling agents form a thioether
rather than a disulfide linkage. Many of these thio-ether-forming
agents are commercially available and include reactive esters of
6-maleimidocaproic acid, 2-bromoacetic acid, and 2-iodoacetic acid,
4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxyl
groups can be activated by combining them with succinimide or
1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt.
[0046] Immunogenic tau peptides of the present invention can be
synthesized by solid phase peptide synthesis or recombinant
expression systems. Automatic peptide synthesizers are commercially
available from numerous suppliers, such as Applied Biosystems
(Foster City, Calif.). Recombinant expression systems can include
bacteria, such as E. coli, yeast, insect cells, or mammalian cells.
Procedures for recombinant expression are described by Sambrook et
al., Molecular Cloning: A Laboratory Manual (C.S.H.P. Press, NY 2d
ed., 1989), which is hereby incorporated by reference in its
entirety.
[0047] The immunogenic tau peptides of the present invention can be
administered alone or in combination with other immunogenic tau
peptides of the present invention to a subject in need. In one
embodiment, an immunogenic tau peptide of the present invention is
administered in combination with one or more immunogenic tau
peptides shown in Table 3 below as disclosed in U.S. Patent
Application Publication No. 20080050383 to Sigurdsson, which is
hereby incorporated by reference in its entirety. The names of the
peptides in Table 3 correspond to the amino acid position of these
peptides within the longest isoform of the tau protein having the
amino acid sequence of SEQ ID NO:1. Amino acid residues of each
sequence which are phosphorylated are shown in bold and marked with
asterisks.
TABLE-US-00004 TABLE 3 Immunogenic Tau Peptide Sequences for
Combined Administration SEQ ID NO: NAME SEQUENCE SEQ ID NO: 81 Tau
133-162 DGTGSDDKKAKGADGKTKIATPRGAAPPGQ SEQ ID NO: 82 Tau 379-408
RENAKAKTDHGAEIVYKS*PVVSGDTS*PRHL [P-Ser.sub.396,404] SEQ ID NO: 83
Tau 192-221 GDRSGYSS*PGS*PGT*PGSRSRT*PS*LPTPPTR
[P-Ser.sub.199,202,214,-Thr.sub.205,212] SEQ ID NO: 84 Tau221-250
REPKKVAVVRT*PPKS*PSSAKSRLQTAPVPM [P-Thr.sub.231,-Ser.sub.235] SEQ
ID NO: 85 Tau184-213 SSGEPPKSGDRSQYSSPGSPGTPGSRSRT SEQ ID NO: 86
Tau1-30 [P-Tyr.sub.18,29] MAEPRQEFEVMEDHAGTY*GLGDRKDQGGY*T SEQ ID
NO: 87 Tau30-60 TMHQDQEGDTDAGLKESPLQTPTEDGSEEPG SEQ ID NO: 88
Tau60-90 GSETSDAKSTPTAEDVTAPLVDEGAPGKQAA SEQ ID NO: 89 Tau90-120
AAQPHTEIPEGTTAEEAGIGDTPSLEDEAAG SEQ ID NO: 90 Tau120-150
GHVTQARMVSKSKDGTGSDDKKAKGADGKTK SEQ ID NO: 91 Tau150-180
[P-Thr.sub.175] KIATPRGAAPPGQKGQANATRIPAKT*PPAPK SEQ ID NO: 92
Tau180-210 [P-Thr.sub.181,205, -
KT*PPS*S*GEPPKSGDRSGY*S*S*PGS*PGT*PGS*RS
Ser.sub.184,185,198,199,202,208, -Tyr.sub.197] SEQ ID NO: 93
Tau210-240 [P-Thr.sub.212,217,231, -
SRT*PS*LPT*PPTREPKKVAVVRT*PPKS*PS*S*AK Ser.sub.214,235,237,238] SEQ
ID NO: 94 Tau240-270 [P-Ser.sub.262]
KSRLQTAPVPMPDLKNVKSKIGS*TENLKHQP SEQ ID NO: 95 Tau270-300
[P-Ser.sub.293] PGGGKVQIINKKLDLSNVQSKCGS*KDNIKHV SEQ ID NO: 96
Tau300-330 [P-Tyr.sub.310, Ser.sub.324]
VPGGGSVQIVY*KPVDLSKVTSKCGS*LGNIHH SEQ ID NO: 97 Tau330-360
[P-Ser.sub.356] HKPGGGQVEVKSEKLDFKDRVQSKIGS*LDNI SEQ ID NO: 98
Tau360-390 ITHVPGGGNKKIETHKLTFRENAKAKTDHGA SEQ ID NO: 99 Tau390-420
[P- AEIVY*KS*PVVS*GDT*S*PRHLS*NVS*S*TGSIDMV Tyr.sub.394,
Ser.sub.396,400,404,409,412,413, Thr.sub.403] SEQ ID Tau411-441
[P-Ser.sub.412,413,422] VS*S*TGSIDMVDS*PQLATLADEVSASLAKQGL NO:
100
[0048] The immunogenic tau peptides of the present invention can be
administered in combination with a suitable adjuvant to achieve the
desired immune response in the subject. Suitable adjuvants can be
administered before, after, or concurrent with administration of
the immunogenic tau peptide of the present invention. Preferred
adjuvants augment the intrinsic response to an immunogen without
causing conformational changes in the immunogen that affect the
qualitative form of the response.
[0049] A preferred class of adjuvants is the aluminum salts (alum),
such as aluminum hydroxide, aluminum phosphate, and aluminum
sulfate. Such adjuvants can be used with or without other specific
immunostimulating agents, such as 3 De-O-acylated monophosphoryl
lipid A (MPL) or 3-DMP, polymeric or monomeric amino acids, such as
polyglutamic acid or polylysine. Such adjuvants can be used with or
without other specific immunostimulating agents, such as muramyl
peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine
(thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'
dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE),
N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy
propylamide (DTP-DPP) Theramide.TM.), or other bacterial cell wall
components. Oil-in-water emulsions include MF59 (see WO 90/14837 to
Van Nest et al., which is hereby incorporated by reference in its
entirety), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85
(optionally containing various amounts of MTP-PE) formulated into
submicron particles using a microfluidizer; SAF, containing 10%
Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and
thr-MDP, either microfluidized into a submicron emulsion or
vortexed to generate a larger particle size emulsion; and the
Ribi.TM. adjuvant system (RAS) (Ribi ImmunoChem, Hamilton, Mont.)
containing 2% squalene, 0.2% Tween 80, and one or more bacterial
cell wall components selected from the group consisting of
monophosphoryllipid A (MPL), trehalose dimycolate (TDM), and cell
wall skeleton (CWS), preferably MPL+CWS (Detox.TM.). Other
adjuvants include Complete Freund's Adjuvant (CFA), Incomplete
Freund's Adjuvant (IFA), and cytokines, such as interleukins (IL-1,
IL-2, and IL-12), macrophage colony stimulating factor (M-CSF), and
tumor necrosis factor (TNF).
[0050] The choice of an adjuvant depends on the stability of the
immunogenic formulation containing the adjuvant, the route of
administration, the dosing schedule, the efficacy of the adjuvant
for the species being vaccinated, and, in humans, a
pharmaceutically acceptable adjuvant is one that has been approved
or is approvable for human administration by pertinent regulatory
bodies. For example, alum, MPL or Incomplete Freund's adjuvant
(Chang et al., Advanced Drug Delivery Reviews 32:173-186 (1998),
which is hereby incorporated by reference in its entirety) alone or
optionally all combinations thereof are suitable for human
administration.
[0051] Another aspect of the present invention relates to a
pharmaceutical composition containing one or more of the
immunogenic tau peptides described supra and a pharmaceutical
carrier (describe infra). The pharmaceutical composition may
contain a mixture of the same immunogenic tau peptide.
Alternatively, the pharmaceutical composition contains a mixture of
one or more different immunogenic tau peptides of the present
invention. In a preferred embodiment, pharmaceutical compositions
of the present invention contain one or more suitable adjuvants as
described supra.
[0052] In another embodiment of the present invention, an antibody
recognizing one or more of the immunogenic tau epitopes of the
present invention is administered to a subject in need. Suitable
antibodies of the present invention encompass any immunoglobulin
molecule that specifically binds to an immunogenic tau epitope
comprising any one of amino acid sequences of SEQ ID NOs: 2-75 and
101-103. In a preferred embodiment, an antibody of the present
invention recognizes and binds to an epitope specific for the
pathological form of tau and has little to no crossreactivity with
the normal tau protein or a non-tau protein.
[0053] As described herein, monoclonal antibodies recognizing the
immunogenic tau epitopes comprising SEQ ID NO:13 (Tau 386-408
[P-Ser.sub.396,404]) and SEQ ID NO:12 (Tau 260-271 [P-Ser.sub.262])
have been generated. These antibodies are phospho-specific and,
therefore, specific for the pathological tau forms having little to
no crossreactivity to the normal tau protein.
[0054] In addition to the antibodies recognizing phosphorylated
pathological epitopes of the tau protein, the present invention is
also directed to antibodies that preferentially recognize
pathological tau fragments involved in promoting neuronal toxicity
and/or seeding tau aggregation. For example, caspase cleavage of
tau, preferentially at aspartate residue 421 (D421) of the tau
protein, creates a truncated molecule that colocalizes with tangles
and correlates with the progression in Alzheimer's disease and in
animal models of tauopathy (see Calignon et al., "Caspase
Activation Precedes and Leads to Tangles," Nature 464:1201-1205
(2010), which is hereby incorporated by reference in its entirety).
An antibody directed to the free D421 end of the cleaved tau
protein would be specific for, and facilitate the removal of,
pathological tau but not normal tau. Accordingly, the present
invention is directed to an antibody, preferably a monoclonal
antibody, directed to D421 on the free C-terminus of a cleaved
pathological tau protein, that is not present in the normal tau
protein. In one embodiment of the present invention, the antibody
is generated using the methods described herein with an immunogenic
tau peptide comprising an amino acid sequence of HLSNVSSTGSIDMVD
(SEQ ID NO:101).
[0055] Truncation of tau at glutamic acid residue 391 (E391) is
also associated with neurofibrillary tangle formation in the brains
of Alzheimer's disease patients (Basurto-Islas et al.,
"Accumulation of Aspartic Acid.sup.421- and Glutamic
Acid.sup.391--Cleaved Tau in Neurofibrillary Tangles Correlates
with Progression in Alzheimer Disease," J Neuropathol Exp Neurol
67:470-483 (2008), which is hereby incorporated by reference in its
entirety). Accordingly, the present invention is also directed to
an antibody, preferably a monoclonal antibody, directed to E391 on
the free C-terminus of a cleaved pathological tau protein, that is
not present in the normal tau protein. In one embodiment of the
present invention, the antibody is generated using the methods
described herein with an immunogenic tau peptide comprising an
amino acid sequence of RENAKAKTDHGAE (SEQ ID NO:102)
[0056] Calpain-1 also mediates the cleavage of tau, generating a
toxic 17 kDa tau fragment that promotes A.beta.-induced
neurotoxicity (Park et al., "The Generation of a 17 kDa Neurotoxic
Fragment: An Alternative Mechanism by which Tau Mediates
.beta.-Amyloid-Induced Neurodegeneration," J Neurosci
25(22):5365-75 (2005), which is hereby incorporated by reference in
its entirety). Accordingly, an embodiment of the present invention
is also directed to an antibody, preferably a monoclonal antibody,
specifically recognizing the free N- and/or free C-terminus of this
toxic tau fragment, but not the normal tau protein, comprising
amino acid residues 45-230 of tau (SEQ ID NO:1) shown as SEQ ID
NO:103 below.
TABLE-US-00005 Glu Ser Pro Leu Gln Thr Pro Thr Glu Asp Gly Ser Glu
Glu Pro Gly 1 5 10 15 Ser Glu Thr Ser Asp Ala Lys Ser Thr Pro Thr
Ala Glu Asp Val Thr 20 25 30 Ala Pro Leu Val Asp Glu Gly Ala Pro
Gly Lys Gln Ala Ala Ala Gln 35 40 45 Pro His Thr Glu Ile Pro Glu
Gly Thr Thr Ala Glu Glu Ala Gly Ile 50 55 60 Gly Asp Thr Pro Ser
Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln 65 70 75 80 Ala Arg Met
Val Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys 85 90 95 Lys
Ala Lys Gly Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly 100 105
110 Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro
115 120 125 Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly
Glu Pro 130 135 140 Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro
Gly Ser Pro Gly 145 150 155 160 Thr Pro Gly Ser Arg Ser Arg Thr Pro
Ser Leu Pro Thr Pro Pro Thr 165 170 175 Arg Glu Pro Lys Lys Val Ala
Val Val Arg 180 185
[0057] As used herein, the term "antibody" includes intact
immunoglobulins derived from natural sources or from recombinant
sources, as well as immunoreactive portions (i.e., antigen binding
portions) of intact immunoglobulins. The antibodies of the present
invention may exist in a variety of forms including, for example,
polyclonal antibodies, monoclonal antibodies, intracellular
antibodies ("intrabodies"), antibody fragments (e.g., Fv, Fab and
F(ab)2), as well as single chain antibodies (scFv), chimeric
antibodies and humanized antibodies (Ed Harlow and David Lane,
USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor
Laboratory Press, 1999); Houston et al., "Protein Engineering of
Antibody Binding Sites: Recovery of Specific Activity in an
Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia
coli," Proc Natl Acad Sci USA 85:5879-5883 (1988); Bird et al,
"Single-Chain Antigen-Binding Proteins," Science 242:423-426
(1988)).
[0058] Methods for monoclonal antibody production may be carried
out using the techniques described herein or others well-known in
the art (MONOCLONAL ANTIBODIES--PRODUCTION, ENGINEERING AND
CLINICAL APPLICATIONS (Mary A. Ritter and Heather M. Ladyman eds.,
1995), which is hereby incorporated by reference in its entirety).
Generally, the process involves obtaining immune cells
(lymphocytes) from the spleen of a mammal which has been previously
immunized with the antigen of interest (i.e., an immunogenic tau
peptide) either in vivo or in vitro. Exemplary tau peptides are
described supra. For generating monoclonal antibodies using the tau
peptides of SEQ ID NOs: 2-75 or tau peptides of SEQ ID NOs:
101-103, a cysteine residue may be added to the N- or C-terminus of
each sequence to facilitate linkage of a carrier protein that will
enhance antibody production upon immunization. Suitable carrier
proteins include, without limitation keyhole limpet hemocyanine,
blue carrier immunogenic protein (derived from Concholepas
concholepas), bovine serum albumin (BSA), ovalbumin, and cationized
BSA.
[0059] The antibody-secreting lymphocytes are fused with myeloma
cells or transformed cells, which are capable of replicating
indefinitely in cell culture, thereby producing an immortal,
immunoglobulin-secreting cell line. Fusion with mammalian myeloma
cells or other fusion partners capable of replicating indefinitely
in cell culture is achieved by standard and well-known techniques,
for example, by using polyethylene glycol (PEG) or other fusing
agents (Milstein and Kohler, "Derivation of Specific
Antibody-Producing Tissue Culture and Tumor Lines by Cell Fusion,"
Eur J Immunol 6:511 (1976), which is hereby incorporated by
reference in its entirety). The immortal cell line, which is
preferably murine, but may also be derived from cells of other
mammalian species, is selected to be deficient in enzymes necessary
for the utilization of certain nutrients, to be capable of rapid
growth, and have good fusion capability. The resulting fused cells,
or hybridomas, are cultured, and the resulting colonies screened
for the production of the desired monoclonal antibodies. Colonies
producing such antibodies are cloned, and grown either in vivo or
in vitro to produce large quantities of antibody.
[0060] Alternatively, monoclonal antibodies can be made using
recombinant DNA methods as described in U.S. Pat. No. 4,816,567 to
Cabilly et al, which is hereby incorporated by reference in its
entirety. The polynucleotides encoding a monoclonal antibody are
isolated from mature B-cells or hybridoma cells, for example, by
RT-PCR using oligonucleotide primers that specifically amplify the
genes encoding the heavy and light chains of the antibody. The
isolated polynucleotides encoding the heavy and light chains are
then cloned into suitable expression vectors, which when
transfected into host cells such as E. coli cells, simian COS
cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do
not otherwise produce immunoglobulin protein, monoclonal antibodies
are generated by the host cells. Also, recombinant monoclonal
antibodies or fragments thereof of the desired species can be
isolated from phage display libraries (McCafferty et al., "Phage
Antibodies: Filamentous Phage Displaying Antibody Variable
Domains," Nature 348:552-554 (1990); Clackson et al., "Making
Antibody Fragments using Phage Display Libraries," Nature
352:624-628 (1991); and Marks et al., "By-Passing Immunization.
Human Antibodies from V-Gene Libraries Displayed on Phage," J. Mol.
Biol. 222:581-597 (1991), which are hereby incorporated by
reference in their entirety).
[0061] The polynucleotide(s) encoding a monoclonal antibody can
further be modified using recombinant DNA technology to generate
alternative antibodies. For example, the constant domains of the
light and heavy chains of a mouse monoclonal antibody can be
substituted for those regions of a human antibody to generate a
chimeric antibody. Alternatively, the constant domains of the light
and heavy chains of a mouse monoclonal antibody can be substituted
for a non-immunoglobulin polypeptide to generate a fusion antibody.
In other embodiments, the constant regions are truncated or removed
to generate the desired antibody fragment of a monoclonal antibody.
Furthermore, site-directed or high-density mutagenesis of the
variable region can be used to optimize specificity and affinity of
a monoclonal antibody.
[0062] The monoclonal antibody of the present invention can be a
humanized antibody. Humanized antibodies are antibodies that
contain minimal sequences from non-human (e.g. murine) antibodies
within the variable regions. Such antibodies are used
therapeutically to reduce antigenicity and human anti-mouse
antibody responses when administered to a human subject.
[0063] An antibody can be humanized by substituting the
complementarity determining region (CDR) of a human antibody with
that of a non-human antibody (e.g. mouse, rat, rabbit, hamster,
etc.) having the desired specificity, affinity, and capability
(Jones et al., "Replacing the Complementarity-Determining Regions
in a Human Antibody With Those From a Mouse," Nature 321:522-525
(1986); Riechmann et al., "Reshaping Human Antibodies for Therapy,"
Nature 332:323-327 (1988); Verhoeyen et al., "Reshaping Human
Antibodies: Grafting an Antilysozyme Activity," Science
239:1534-1536 (1988), which are hereby incorporated by reference in
their entirety). The humanized antibody can be further modified by
the substitution of additional residues either in the Fv framework
region and/or within the replaced non-human residues to refine and
optimize antibody specificity, affinity, and/or capability.
[0064] Human antibodies can be produced using various techniques
known in the art. Immortalized human B lymphocytes immunized in
vitro or isolated from an immunized individual that produce an
antibody directed against a target antigen can be generated (See
e.g. Reisfeld et al., MONOCLONAL ANTIBODIES AND CANCER THERAPy 77
(Alan R. Liss ed., 1985) and U.S. Pat. No. 5,750,373 to Garrard,
which are hereby incorporated by reference in their entirety).
Alternatively, the human antibody can be selected from a phage
library, where that phage library expresses human antibodies
(Vaughan et al., "Human Antibodies with Sub-Nanomolar Affinities
Isolated from a Large Non-immunized Phage Display Library," Nature
Biotechnology, 14:309-314 (1996); Sheets et al., "Efficient
Construction of a Large Nonimmune Phage Antibody Library: The
Production of High-Affinity Human Single-Chain Antibodies to
Protein Antigens," Proc. Natl. Acad. Sci. U.S.A. 95:6157-6162
(1998); Hoogenboom et al., "By-passing Immunization. Human
Antibodies From Synthetic Repertoires of Germline VH Gene Segments
Rearranged In Vitro," J Mol Biol 227:381-8 (1992); Marks et al.,
"By-passing Immunization. Human Antibodies from V-gene Libraries
Displayed on Phage," J Mol Biol 222:581-97 (1991), which are hereby
incorporated by reference in their entirety). Human antibodies can
also be made in transgenic mice containing human immunoglobulin
loci that are capable upon immunization of producing the full
repertoire of human antibodies in the absence of endogenous
immunoglobulin production. This approach is described in U.S. Pat.
No. 5,545,807 to Surani et al.; U.S. Pat. No. 5,545,806 to Lonberg
et al.; U.S. Pat. No. 5,569,825 to Lonberg et al.; U.S. Pat. No.
5,625,126 to Lonberg et al.; U.S. Pat. No. 5,633,425 to Lonberg et
al.; and U.S. Pat. No. 5,661,016 to Lonberg et al., which are
hereby incorporated by reference in their entirety
[0065] Procedures for raising polyclonal antibodies are also well
known in the art. Typically, such antibodies can be raised by
administering the peptide containing the epitope of interest (i.e.
any tau peptide selected from the group consisting of SEQ ID NOs:
2-75 or SEQ ID NOs: 101-103) subcutaneously to New Zealand white
rabbits which have been bled to obtain pre-immune serum. The
antigens can be injected in combination with an adjuvant. The
rabbits are bled approximately every two weeks after the first
injection and periodically boosted with the same antigen three
times every six weeks. Polyclonal antibodies are recovered from the
serum by affinity chromatography using the corresponding antigen to
capture the antibody. This and other procedures for raising
polyclonal antibodies are disclosed in Ed Harlow and David Lane,
USING ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor
Laboratory Press, 1988), which is hereby incorporated by reference
in its entirety.
[0066] In addition to whole antibodies, the present invention
encompasses binding portions of such antibodies. Such binding
portions include the monovalent Fab fragments, Fv fragments (e.g.,
single-chain antibody, scFv), and single variable V.sub.H and
V.sub.L domains, and the bivalent F(ab').sub.2 fragments, Bis-scFv,
diabodies, triabodies, minibodies, etc. These antibody fragments
can be made by conventional procedures, such as proteolytic
fragmentation procedures, as described in James Goding, MONOCLONAL
ANTIBODIES: PRINCIPLES AND PRACTICE 98-118 (Academic Press, 1983)
and Ed Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL (Cold
Spring Harbor Laboratory, 1988), which are hereby incorporated by
reference in their entirety, or other methods known in the art.
[0067] Also suitable for use in the present invention are antibody
fragments engineered to bind to intracellular proteins, i.e.
intrabodies. Intrabodies directed to an immunogenic tau epitope
comprising any one of SEQ ID NOs: 2-75 of SEQ ID NOs: 101-103 can
prevent pathological tau aggregation and accumulation within
neurons or glial cells and/or facilitate aggregate clearance. The
application of intrabody technology for the treatment of
neurological disorders, including tauopathies, is reviewed in
Miller et al., "Intrabody Applications in Neurological Disorders:
Progress and Future Prospects," Mol Therapy 12:394-401 (2005),
which is hereby incorporated by reference in its entirety.
[0068] Intrabodies are generally obtained by selecting a single
variable domain from variable regions of an antibody having two
variable domains (i.e., a heterodimer of a heavy chain variable
domain and a light chain variable domain). Single chain Fv
fragments, Fab fragments, ScFv-Ck fusion proteins, single chain
diabodies, V.sub.H-C.sub.H1 fragments, and even whole IgG molecules
are suitable formats for intrabody development (Kontermann R. E.,
"Intrabodies as Therapeutic Agents," Methods 34:163-70 (2004),
which is here by incorporated by reference in its entirety).
[0069] Intrabodies having antigen specificity for a pathological
tau protein epitope can be obtained from phage display, yeast
surface display, or ribosome surface display. Methods for producing
libraries of intrabodies and isolating intrabodies of interest are
further described in U.S. Published Patent Application No.
20030104402 to Zauderer and U.S. Published Patent Application No.
20050276800 to Rabbitts, which are hereby incorporated by reference
in their entirety. Methods for improving the stability and affinity
binding characteristics of intrabodies are described in
WO2008070363 to Zhenping and Contreras-Martinez et al.,
"Intracellular Ribosome Display via SecM Translation Arrest as a
Selection for Antibodies with Enhanced Cytosolic Stability," J Mol
Biol 372(2):513-24 (2007), which are hereby incorporated by
reference in their entirety.
[0070] It may further be desirable, especially in the case of
antibody fragments, to modify the antibody in order to increase its
serum half-life. This can be achieved, for example, by
incorporation of a salvage receptor binding epitope into the
antibody fragment by mutation of the appropriate region in the
antibody fragment or by incorporating the epitope into a peptide
tag that is then fused to the antibody fragment at either end or in
the middle (e.g., by DNA or peptide synthesis).
[0071] Antibody mimics are also suitable for use in accordance with
the present invention. A number of antibody mimics are known in the
art including, without limitation, those known as monobodies, which
are derived from the tenth human fibronectin type III domain
(.sup.10Fn3) (Koide et al., "The Fibronectin Type III Domain as a
Scaffold for Novel Binding Proteins," J Mol Biol 284:1141-1151
(1998); Koide et al., "Probing Protein Conformational Changes in
Living Cells by Using Designer Binding Proteins: Application to the
Estrogen Receptor," Proc Natl Acad Sci USA 99:1253-1258 (2002),
each of which is hereby incorporated by reference in its entirety),
and those known as affibodies, which are derived from the stable
alpha-helical bacterial receptor domain Z of staphylococcal protein
A (Nord et al., "Binding Proteins Selected from Combinatorial
Libraries of an alpha-helical Bacterial Receptor Domain," Nature
Biotechnol 15(8):772-777 (1997), which is hereby incorporated by
reference in its entirety).
[0072] The present invention is further directed to pharmaceutical
compositions containing the one or more antibodies recognizing the
immunogenic tau peptides of the present invention as described
supra. This pharmaceutical composition may contain a mixture of the
same antibodies recognizing the same tau epitope. Alternatively,
the pharmaceutical composition may contain a mixture of one or more
antibodies recognizing one or more different tau epitopes. The
pharmaceutical composition of the present invention further
contains a pharmaceutically acceptable carrier or other
pharmaceutically acceptable components as described infra.
[0073] The pharmaceutical compositions of the present invention
containing the immunogenic tau peptides or antibodies recognizing
the immunogenic tau peptides, contain, in addition to the active
therapeutic agent, a variety of other pharmaceutically acceptable
components (see Remington's Pharmaceutical Science (15th ed., Mack
Publishing Company, Easton, Pa., 1980), which is hereby
incorporated by reference in its entirety). The preferred
formulation of the pharmaceutical composition depends on the
intended mode of administration and therapeutic application. The
compositions can include pharmaceutically-acceptable, non-toxic
carriers or diluents, which are defined as vehicles commonly used
to formulate pharmaceutical compositions for animal or human
administration. The diluent is selected so as not to affect the
biological activity of the combination. Examples of such diluents
are distilled water, physiological phosphate-buffered saline,
Ringer's solutions, dextrose solution, and Hank's solution. In
addition, the pharmaceutical composition or formulation may also
include other carriers, adjuvants, or nontoxic, nontherapeutic,
non-immunogenic stabilizers, and the like.
[0074] Pharmaceutical compositions can also include large, slowly
metabolized macromolecules, such as proteins, polysaccharides like
chitosan, polylactic acids, polyglycolic acids and copolymers
(e.g., latex functionalized sepharose, agarose, cellulose, and the
like), polymeric amino acids, amino acid copolymers, and lipid
aggregates (e.g., oil droplets or liposomes). Additionally, these
carriers can function as immunostimulating agents (i.e.,
adjuvants).
[0075] The pharmaceutical compositions of the present invention can
further include a suitable delivery vehicle. Suitable delivery
vehicles include, but are not limited to viruses, bacteria,
biodegradable microspheres, microparticles, nanoparticles,
liposomes, collagen minipellets, and cochleates.
[0076] In one embodiment of the present invention, the delivery
vehicle is a virus or bacteria and the immunogenic tau peptide is
presented by a virus or bacteria as part of an immunogenic
composition. In accordance with this embodiment of the invention, a
nucleic acid molecule encoding the immunogenic peptide is
incorporated into a genome or episome of the virus or bacteria.
Optionally, the nucleic acid molecule is incorporated in such a
manner that the immunogenic peptide is expressed as a secreted
protein or as a fusion protein with an outer surface protein of a
virus or a transmembrane protein of bacteria so that the peptide is
displayed. Viruses or bacteria used in such methods should be
nonpathogenic or attenuated. Suitable viruses include adenovirus,
HSV, Venezuelan equine encephalitis virus and other alpha viruses,
vesicular stomatitis virus, and other rhabdo viruses, vaccinia and
fowl pox. Suitable bacteria include Salmonella and Shigella. Fusion
of an immunogenic peptide to HBsAg of HBV is particularly
suitable.
[0077] In another embodiment of the present invention, the
pharmaceutical composition contains a liposome delivery vehicle.
Liposomes are vesicles comprised of one or more concentrically
ordered lipid bilayers which encapsulate an aqueous phase. An
immunogenic tau peptide or antibody raised against an immunogenic
tau peptide of the present invention can be surface bound,
encapsulated, or associated with the membrane of the liposome
vehicle. Various types of liposomes suitable for vaccine delivery
of the tau peptides are known in the art (see e.g., Hayashi et al.,
"A Novel Vaccine Delivery System Using Immunopotentiating Fusogenic
Liposomes," Biochem Biophys Res Commun 261(3):824-28 (1999) and
U.S. Patent Publication No. 20070082043 to Michaeli et al., which
are hereby incorporated by reference in their entirety). Other
methods for preparing liposomes for use in the present invention
include those disclosed in Bangham et al., "Diffusion of Univalent
Ions Across the Lamellae of Swollen Phospholipids," J. Mol. Biol.
13:238-52 (1965); U.S. Pat. No. 5,653,996 to Hsu; U.S. Pat. No.
5,643,599 to Lee et al.; U.S. Pat. No. 5,885,613 to Holland et al.;
U.S. Pat. No. 5,631,237 to Dzau & Kaneda; and U.S. Pat. No.
5,059,421 to Loughrey et al., which are hereby incorporated by
reference in their entirety.
[0078] In another embodiment of the present invention, a nucleic
acid molecule encoding an immunogenic tau peptide or a tau antibody
of the present invention is administered using a gene therapy
delivery system. Suitable gene therapy vectors include, without
limitation, adenoviral vectors, adeno-associated viral vectors,
retroviral vectors, lentiviral vectors, and herpes viral
vectors.
[0079] Adenoviral viral vector delivery vehicles can be readily
prepared and utilized as described in Berkner, "Development of
Adenovirus Vectors for the Expression of Heterologous Genes,"
Biotechniques 6:616-627 (1988) and Rosenfeld et al.,
"Adenovirus-Mediated Transfer of a Recombinant Alpha 1-Antitrypsin
Gene to the Lung Epithelium In Vivo," Science 252:431-434 (1991),
WO 93/07283 to Curiel et al., WO 93/06223 to Perricaudet et al.,
and WO 93/07282 to Curiel et al., which are hereby incorporated by
reference in their entirety. Adeno-associated viral delivery
vehicles can be constructed and used to deliver a nucleic acid
encoding a tau antibody of the present invention to cells as
described in Shi et al., "Therapeutic Expression of an Anti-Death
Receptor-5 Single-Chain Fixed Variable Region Prevents Tumor Growth
in Mice," Cancer Res. 66:11946-53 (2006); Fukuchi et al.,
"Anti-A.beta. Single-Chain Antibody Delivery via Adeno-Associated
Virus for Treatment of Alzheimer's Disease," Neurobiol. Dis.
23:502-511 (2006); Chatterjee et al., "Dual-Target Inhibition of
HIV-1 In Vitro by Means of an Adeno-Associated Virus Antisense
Vector," Science 258:1485-1488 (1992); Ponnazhagan et al.,
"Suppression of Human Alpha-Globin Gene Expression Mediated by the
Recombinant Adeno-Associated Virus 2-Based Antisense Vectors," J.
Exp. Med. 179:733-738 (1994); and Zhou et al., "Adeno-associated
Virus 2-Mediated Transduction and Erythroid Cell-Specific
Expression of a Human Beta-Globin Gene," Gene Ther. 3:223-229
(1996), which are hereby incorporated by reference in their
entirety. In vivo use of these vehicles is described in Flotte et
al., "Stable in Vivo Expression of the Cystic Fibrosis
Transmembrane Conductance Regulator With an Adeno-Associated Virus
Vector," Proc. Nat'l. Acad. Sci. 90:10613-10617 (1993) and Kaplitt
et al., "Long-Term Gene Expression and Phenotypic Correction Using
Adeno-Associated Virus Vectors in the Mammalian Brain," Nature
Genet. 8:148-153 (1994), which are hereby incorporated by reference
in their entirety. Additional types of adenovirus vectors are
described in U.S. Pat. No. 6,057,155 to Wickham et al.; U.S. Pat.
No. 6,033,908 to Bout et al.; U.S. Pat. No. 6,001,557 to Wilson et
al.; U.S. Pat. No. 5,994,132 to Chamberlain et al.; U.S. Pat. No.
5,981,225 to Kochanek et al.; U.S. Pat. No. 5,885,808 to Spooner et
al.; and U.S. Pat. No. 5,871,727 to Curiel, which are hereby
incorporated by reference in their entirety.
[0080] Retroviral vectors which have been modified to form
infective transformation systems can also be used to deliver
nucleic acid molecules encoding a desired peptide or antibody to a
target cell. One such type of retroviral vector is disclosed in
U.S. Pat. No. 5,849,586 to Kriegler et al., which is hereby
incorporated by reference.
[0081] Gene therapy vectors carrying a nucleic acid molecule
encoding the immunogenic tau peptide or tau antibody are
administered to a subject by, for example, intravenous injection,
local administration (U.S. Pat. No. 5,328,470 to Nabel et al.,
which is hereby incorporated by reference in its entirety) or by
stereotactic injection (see e.g., Chen et al., "Gene Therapy for
Brain Tumors: Regression of Experimental Gliomas by Adenovirus
Mediated Gene Transfer In Vivo," Proc. Nat'l. Acad. Sci. USA
91:3054-3057 (1994), which is hereby incorporated by reference in
its entirety). The pharmaceutical preparation of the gene therapy
vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is imbedded.
[0082] In carrying out the methods of the present invention, it is
preferable to select a subject having or at risk of having
Alzheimer's disease or other tauopathy, a subject having tau
aggregates in the brain, or a subject exhibiting a tangle related
behavioral phenotype prior to administering the immunogenic
peptides or antibodies of the present invention. Subjects amenable
to treatment include individuals at risk of disease but not showing
symptoms, as well as patients presently showing symptoms. In the
case of Alzheimer's disease, virtually anyone is at risk of
suffering from Alzheimer's disease. Therefore, the present methods
can be administered prophylactically to the general population
without the need for any assessment of the risk of the subject
patient. The present methods are especially useful for individuals
who do have a known genetic risk of Alzheimer's disease. Such
individuals include those having relatives who have experienced
this disease, and those whose risk is determined by analysis of
genetic or biochemical markers. Genetic markers of risk toward
Alzheimer's disease include mutations in the APP gene, particularly
mutations at position 717 and positions 670 and 671 referred to as
the Hardy and Swedish mutations, respectively. Other markers of
risk include mutations in the presenilin genes, PS1 and PS2, and
ApoE4 gene, a family history of AD, and hypercholesterolemia or
atherosclerosis. Individuals presently suffering from Alzheimer's
disease can be recognized from characteristic dementia by the
presence of risk factors described above. In addition, a number of
diagnostic tests are available for identifying individuals who have
AD. These include measurement of CSF tau and A.beta.42 levels.
Elevated tau and decreased A.beta.42 levels signify the presence of
AD. Individuals suffering from Alzheimer's disease can also be
diagnosed by Alzheimer's Disease and Related Disorders Association
criteria.
[0083] In asymptomatic patients, treatment can begin at any age
(e.g., 10, 20, 30 years of age). Usually, however, it is not
necessary to begin treatment until a patient reaches 40, 50, 60, or
70 years of age. Treatment typically entails multiple dosages over
a period of time. Treatment can be monitored by assaying antibody,
or activated T-cell or B-cell responses to the therapeutic agent
over time. If the response falls, a booster dosage is indicated. In
the case of potential Down's syndrome patients, treatment can begin
antenatally by administering therapeutic agent to the mother or
shortly after birth.
[0084] In prophylactic applications, pharmaceutical compositions
containing the immunogenic tau peptides are administered to a
patient susceptible to, or otherwise at risk of, Alzheimer's
disease or other tauopathy in an amount sufficient to eliminate or
reduce the risk, lessen the severity, or delay the outset of the
disease, including biochemical, histologic and/or behavioral
symptoms of the disease, its complications and intermediate
pathological phenotypes presented during development of the
disease. In therapeutic applications, compositions containing a tau
antibody are administered to a patient suspected of, or already
suffering from, such a disease in an amount sufficient to cure, or
at least partially arrest, the symptoms of the disease
(biochemical, histologic and/or behavioral), including its
complications and intermediate pathological phenotypes in
development of the disease. In some methods, administration of
agent reduces or eliminates mild cognitive impairment in patients
that have not yet developed characteristic Alzheimer's pathology.
An amount adequate to accomplish therapeutic or prophylactic
treatment is defined as a therapeutically- or
prophylactically-effective dose. In both prophylactic and
therapeutic regimes, agents are usually administered in several
dosages until a sufficient immune response has been achieved.
Typically, the immune response is monitored and repeated dosages
are given if the immune response starts to wane.
[0085] Effective doses of the compositions of the present
invention, for the treatment of the above described conditions vary
depending upon many different factors, including mode of
administration, target site, physiological state of the patient,
other medications administered, and whether treatment is
prophylactic or therapeutic. Treatment dosages need to be titrated
to optimize safety and efficacy. The amount of immunogen depends on
whether adjuvant is also administered, with higher dosages being
required in the absence of adjuvant. The amount of an immunogen for
administration sometimes varies from 1-500 .mu.g per patient and
more usually from 5-500 .mu.g per injection for human
administration. Occasionally, a higher dose of 1-2 mg per injection
is used. Typically about 10, 20, 50, or 100 .mu.g is used for each
human injection. The mass of immunogen also depends on the mass
ratio of immunogenic epitope within the immunogen to the mass of
immunogen as a whole. Typically, 10.sup.-3 to 10.sup.-5 micromoles
of immunogenic epitope are used for each microgram of immunogen.
The timing of injections can vary significantly from once a day, to
once a year, to once a decade. On any given day that a dosage of
immunogen is given, the dosage is greater than 1 .mu.g/patient and
usually greater than 10 .mu.g/patient if adjuvant is also
administered, and greater than 10 .mu.g/patient and usually greater
than 100 .mu.g/patient in the absence of adjuvant. A typical
regimen consists of an immunization followed by booster injections
at time intervals, such as 6 week intervals. Another regimen
consists of an immunization followed by booster injections 1, 2,
and 12 months later. Another regimen entails an injection every two
months for life. Alternatively, booster injections can be on an
irregular basis as indicated by monitoring of immune response.
[0086] For passive immunization with an antibody, the dosage ranges
from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg,
of the host body weight. For example dosages can be 1 mg/kg body
weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.
An exemplary treatment regime entails administration once per every
two weeks or once a month or once every 3 to 6 months. In some
methods, two or more monoclonal antibodies with different binding
specificities are administered simultaneously, in which case the
dosage of each antibody administered falls within the ranges
indicated. Antibody is usually administered on multiple occasions.
Intervals between single dosages can be weekly, monthly, or yearly.
In some methods, dosage is adjusted to achieve a plasma antibody
concentration of 1-1000 .mu.g/ml and in some methods 25-300
.mu.g/ml. Alternatively, antibody can be administered as a
sustained release formulation, in which case less frequent
administration is required. Dosage and frequency vary depending on
the half-life of the antibody in the patient. In general, human
antibodies show the longest half life, followed by humanized
antibodies, chimeric antibodies, and nonhuman antibodies. The
dosage and frequency of administration can vary depending on
whether the treatment is prophylactic or therapeutic. In
prophylactic applications, a relatively low dosage is administered
at relatively infrequent intervals over a long period of time. Some
patients continue to receive treatment for the rest of their lives.
In therapeutic applications, a relatively high dosage at relatively
short intervals is sometimes required until progression of the
disease is reduced or terminated, and preferably until the patient
shows partial or complete amelioration of symptoms of disease.
Thereafter, the patent can be administered a prophylactic
regime.
[0087] Doses for nucleic acids encoding immunogens range from about
10 ng to about 1 g, from about 100 ng to about 100 mg, from about 1
.mu.g to about 10 mg, or from about 30 to about 300 .mu.g DNA per
patient. Doses for infectious viral vectors vary from 10-100, or
more, virions per dose.
[0088] Agents for inducing an immune response can be administered
by parenteral, topical, intravenous, oral, subcutaneous,
intraarterial, intracranial, intraperitoneal, intranasal, or
intramuscular means for prophylactic and/or therapeutic treatment.
The most typical route of administration of an immunogenic agent is
subcutaneous, although other routes can be equally effective. The
next most common route is intramuscular injection. This type of
injection is most typically performed in the arm or leg muscles. In
some cases, it may be desirable to inject the therapeutic agent of
the present invention directly into a particular tissue where
deposits have accumulated, for example intracranial injection.
Intramuscular injection or intravenous infusion is preferred for
administration of antibody. In some methods, particular therapeutic
antibodies are injected directly into the cranium. In some methods,
antibodies are administered as a sustained release composition or
device, such as a Medipad.TM. device (Elan Pharm. Technologies,
Dublin, Ireland).
[0089] Another aspect of the present invention is directed to a
combination therapy where an immunogenic tau peptide or antibody
recognizing an immunogenic tau epitope of the present invention is
administered in combination with agents that are effective for the
prevention or treatment of other conditions or diseases associated
with, or resulting from, the deposition of amyloidogenic proteins
or peptides. Amyloidogenic proteins/peptides subject to deposition
include, without limitation, beta protein precursor, prion and
prion proteins, .alpha.-synuclein, tau, ABri precursor protein,
ADan precursor protein, islet amyloid polypeptide, apolipoprotein
AI, apolipoprotein AII, lyzozyme, cystatin C, gelsolin, atrial
natriuretic factor, calcitonin, keratoepithelin, lactoferrin,
immunoglobulin light chains, transthyretin, A amyloidosis,
.beta.2-microglobulin, immunoglobulin heavy chains, fibrinogen
alpha chains, prolactin, keratin, and medin. Therefore, a
combination therapeutic of the present invention would include an
immunogenic tau peptide or antibody recognizing an immunogenic tau
epitope and an agent or agents targeting one or more of the
aforementioned amyloidogenic proteins or peptides.
[0090] In the case of amyloidogenic diseases such as, Alzheimer's
disease and Down's syndrome, immune modulation to clear
amyloid-beta (A.beta.) deposits is an emerging therapy.
Immunotherapies targeting A.beta. have consistently resulted in
cognitive improvements. It is likely that tau and A.beta.
pathologies are synergistic. Therefore, combination therapy
targeting the clearance of both tau and A.beta. and A.beta.-
-related pathologies at the same time may be more effective than
targeting each individually.
[0091] In the case of Parkinson's Disease and related
neurodegenerative diseases, immune modulation to clear aggregated
forms of the .alpha.-synuclein protein is also an emerging therapy.
A combination therapy which targets the clearance of both tau and
.alpha.-synuclein proteins simultaneously may be more effective
than targeting either protein individually.
[0092] In the case of prion disease and related neurodegenerative
diseases, immune modulation to clear the disease associated form of
the prion protein, PrP.sup.Sc, is an emerging therapy. Therefore, a
combination therapy which targets the clearance of both tau and the
pathological PrP.sup.Sc protein simultaneously may be more
effective than targeting either protein individually.
[0093] Individuals with type-2 diabetes may be more prone to the
development of Alzheimer's disease. Therefore, a combination
therapy which includes an agent targeting the clearance of islet
amyloid polypeptide and an agent preventing the development or
progression of Alzheimer's diseases (i.e., preventing tau
deposition) would have enhanced therapeutic benefit to the
individual.
[0094] Another aspect of the present invention relates to a method
of diagnosing an Alzheimer's disease or other tauopathy in a
subject. This method involves detecting, in the subject, the
presence of pathological tau conformer using a diagnostic reagent,
where the diagnostic reagent is an antibody, or active binding
fragment thereof, of the present invention. As described supra, the
antibody has antigenic specificity for an isolated tau peptide
having an amino acid sequence selected from SEQ ID NOs: 2-75 or SEQ
ID NOs: 101-103. The diagnosis of the Alzheimer's disease or other
tauopathy is based on the detection of a pathological tau conformer
in the subject.
[0095] Detecting the presence of a pathological tau conformer in a
subject using the diagnostic antibody reagent of the present
invention can be achieved by obtaining a biological sample from the
subject (e.g., blood, urine, cerebral spinal fluid), contacting the
biological sample with the diagnostic antibody reagent, and
detecting binding of the diagnostic antibody reagent to a
pathological tau protein conformer in the sample from the subject.
Assays for carrying out the detection of a pathological tau protein
in a biological sample using the diagnostic antibody of the present
invention are well known in the art and include, without
limitation, ELISA, immunohistochemistry, western blot.
[0096] Alternatively, detecting the presence of a pathological tau
protein conformer in a subject using the diagnostic antibody
reagent of the present invention can be achieved using in vivo
imaging techniques. In vivo imaging involves administering to the
subject the diagnostic antibody having antigenic specificity for a
pathological tau peptide or epitope (i.e., SEQ ID NOs: 2-75 and
101-103) and detecting binding of the diagnostic antibody reagent
to the pathological tau protein conformer in vivo. As described
supra, preferred antibodies bind to the pathological tau protein
without binding to non-tau proteins and without binding to the
non-pathological forms of tau.
[0097] Diagnostic antibodies or similar reagents can be
administered by intravenous injection into the body of the patient,
or directly into the brain by intracranial injection. The dosage of
antibody should be within the same ranges as for treatment methods.
Typically, the antibody is labeled, although in some methods, the
primary antibody with affinity for the pathological tau protein is
unlabelled and a secondary labeling agent is used to bind to the
primary antibody. The choice of label depends on the means of
detection. For example, a fluorescent label is suitable for optical
detection. Use of paramagnetic labels is suitable for tomographic
detection without surgical intervention. Radioactive labels can
also be detected using PET or SPECT.
[0098] Diagnosis is performed by comparing the number, size, and/or
intensity of labeled pathological tau conformers, tau aggregates,
and/or neurofibrillary tangles in a sample from the subject or in
the subject, to corresponding baseline values. The base line values
can represent the mean levels in a population of undiseased
individuals. Baseline values can also represent previous levels
determined in the same subject.
[0099] The diagnostic methods described above can also be used to
monitor a subject's response to therapy. In this embodiment,
detecting the presence of pathological tau in a subject is
determined prior to the commencement of treatment. The level of
pathological tau in the subject at this timepoint is used as a
baseline value. At various times during the course of treatment the
detection of pathological tau protein conformers, tau aggregates,
and/or neurofibrillary tangles is repeated, and the measured values
thereafter compared with the baseline values. A decrease in values
relative to baseline signals a positive response to treatment.
Values can also increase temporarily in biological fluids as
pathological tau is being cleared from the brain.
[0100] The present invention is further directed to a kit for
performing the above described diagnostic and monitoring methods.
Typically, such kits contain a diagnostic reagent, preferably the
antibody of the present invention that has antigenic specificity
for a pathological tau peptide (i.e., SEQ ID NOs: 2-75 and
101-103). The kit can also include a detectable label. The
diagnostic antibody itself may contain the detectable label (e.g.,
fluorescent molecule, biotin, etc.) which is directly detectable or
detectable via a secondary reaction (e.g., reaction with
streptavidin). Alternatively, a second reagent containing the
detectable label may be utilized, where the second reagent has
binding specificity for the primary antibody. In a diagnostic kit
suitable for measuring pathological tau protein in a biological
sample, the antibodies of the kit may be supplied prebound to a
solid phase, such as to the wells of a microtiter dish.
[0101] Diagnostic kits of the present invention also include kits
that are useful for detecting antibody production in a subject
following administration of an immunogenic tau peptide of the
present invention. Typically, such kits include a reagent that
contains the antigenic epitope of the antibodies generated by the
subject. The kit also includes a detectable label. In a preferred
embodiment, the label is typically in the form of labeled
anti-idiotypic antibodies. The reagent of the kit can be supplied
prebound to a solid phase, such as to the wells of a microtiter
dish.
[0102] The following examples illustrate various methods for
compositions in the treatment method of the invention. The examples
are intended to illustrate, but in no way limit, the scope of the
invention.
EXAMPLES
Example 1
Peptides
[0103] The peptide immunogens were synthesized at the Keck facility
(Yale University), by the solid-phase technique on a
p-methyl-benzhydrylamine resin, using a Biosearch SAM 2 synthesizer
(Biosearch, Inc., San Rafael, Ca.). The peptides were cleaved from
the resin with HF and then extracted with ether and acetic acid
before lyophilization. Subsequently, the peptides were purified by
HPLC with the use of a reverse-phase support medium
(Delta-Bondapak) on a 0.78.times.30 cm column with a 0-66% linear
gradient of acetonitrile in 0.1% TFA.
Example 2
Animals Used in Studies
[0104] Studies were performed in the transgenic (Tg) JNPL3 P301L
mouse model that develops neurofibrillary tangles in several brain
regions and spinal cord (Taconic, Germantown, N.Y.) (Lewis et al.,
"Neurofibrillary Tangles, Amyotrophy and Progressive Motor
Disturbance in Mice Expressing Mutant (P301L) Tau Protein," Nat
Genet 25:402-405 (2000), which is hereby incorporated by reference
in its entirety). While this model is not ideal for AD, it is an
excellent model to study the consequences of tangle development and
for screening therapy that may prevent the generation of these
aggregates. Another advantage of these animals is the relatively
early onset of pathology. In the homozygous line, behavioral
abnormalities associated with tau pathology can be observed at
least as early as 3 months, but the animals remain relatively
healthy at least until 8 months of age. In other words, at 8
months, the animals ambulate, feed themselves, and can perform the
behavioral tasks sufficiently well to allow the treatment effect to
be monitored.
[0105] In addition to the JNPL3 P301L model, studies were also
carried out using an htau/PS1 (M146L) mouse model (Boutajangout et
al., "Presenilin 1 Mutation Promotes Tau Phosphorylation and
Aggregation in a Novel Alzheimer's Disease Mouse Model,"
Alzheimer's and Dementia 4:T185 (2008), which is hereby
incorporated by reference in its entirety). htau mice express
unmutated human tau protein on a null mouse tau background and
better resembles Alzheimer's tau pathology in the age of onset and
brain distribution (Andorfer et al., "Hyperphosphorylation and
Aggregation of Tau in Mice Expressing Normal Human Tau Isoforms," J
Neurochem 86: 582-90 (2003), which is hereby incorporated by
reference in its entirety). The PS1 model, carrying a mutation
(M146L) in the Presenlin 1 protein, has shown to have increased
A.beta. levels and to promote A.beta. deposition when crossed with
Tg2576 mice (Duff et al., "Increased Amyloid-beta 42(43) in Brains
of Mice Expressing Mutant Presenilin 1," Nature 383:710-713 (1996)
and Holcomb et al., "Accelerated Alzheimer-Type Phenotype in
Transgenic Mice Carrying Both Mutant Amyloid Precursor Protein and
Presenilin 1 Transgenes," Nature Med 4:97-100 (1998), which are
hereby incorporated by reference in their entirety).
[0106] htau mice, expressing all six human isoforms of tau, were
crossed with PS1 (M146L) mice and maintained on a mouse tau
knockout background (htau/PS1/mtau-/-). The PS1 mutation promotes
hyperphosphorylation of tau in this model which leads to more
aggressive tau pathology with earlier onset than in the htau model
(Boutaj angout et al., "Presenilin 1 Mutation Promotes Tau
Phosphorylation and Aggregation in a Novel Alzheimer's Disease
Mouse Model," Alzheimer's and Dementia 4:T185 (2008), which is
hereby incorporated by reference in its entirety).
Example 3
Vaccine Administration
[0107] Phos-tau peptides were mixed with Adju-Phos adjuvant
(Brenntag Biosector, Denmark) at a concentration of 1 mg/ml and the
solution was rotated overnight at 4.degree. C. prior to
administration to allow the peptide to adsorb onto the aluminum
phosphate particles.
[0108] JNPL3 P301L mice received a subcutaneous injection of 100
.mu.l followed by a second injection 2 weeks later and then monthly
thereafter (unless otherwise indicated). Vaccination started at 2-3
months of age and continued until the animals were 8-9 months of
age at which time the animals were perfused and their organs
collected for analysis. The mice went through a battery of
sensorimotor tests at 5-6 months and again at 8-9 months of age
prior to sacrifice. Control mice received the adjuvant alone.
[0109] htau/PS1/mtau-/- mice (n=12) were immunized with the
phosphorylated tau immunogen Tau379-408[P-Ser396,404]. Three
non-immunized control groups were included that received adjuvant
alone. The main control group consisted of identical mice that were
not immunized (htau/PS1 controls; n=16). Other control groups were
htau/PS1 mice that expressed mouse tau (htau/PS l/mtau; n=8) as
well as htau littermates on a mouse tau knockout background (htau
controls; n=10).
[0110] htau/PS1/mtau-/- mice (3-4 months of age) received 100 .mu.g
of the phosphorylated tau derivative intraperitoneally (i.p.) in
alum adjuvant with the first 3 injections every 2 weeks. Subsequent
administration was at monthly intervals. The control groups
received adjuvant alone. At 7-8 months the mice went through
extensive behavioral testing to determine treatment efficacy, and
were subsequently killed for analysis at 8-9 months of age.
Locomotor activity, traverse beam, and rotarod tests were performed
to determine if measured cognitive deficits in the learning and
memory tasks could be attributed to sensorimotor abnormalities.
Cognitive testing was performed using the radial arm maze, the
closed field symmetrical maze, and the object recognition test
(Sigurdsson et al., "An Attenuated Immune Response is Sufficient to
Enhance Cognition in an Alzheimer's Disease Mouse Model Immunized
with Amyloid-beta Derivatives," J Neurosci 24:6277-6282 (2004),
Asuni et al., "Vaccination of Alzheimer's Model Mice with Abeta
Derivative in Alum Adjuvant Reduces Abeta Burden Without
Microhemorrhages." Eur J Neurosci. 24:2530-42 (2006), and Asuni et
al., "Immunotherapy Targeting Pathological Tau Conformers in a
Tangle Mouse Model Reduces Brain Pathology with Associated
Functional Improvements," J Neurosci 27:9115-9129 (2007), which are
hereby incorporated by reference in their entirety).
Example 4
Tau Immunotherapy Generates a Robust Antibody Response
[0111] The mice were bled prior to the commencement of the study
(T0), a week following the third injection, periodically
thereafter, and at sacrifice (Tf). The antibody response to the
vaccine was determined by dilution of plasma (1:200 unless
otherwise indicated) using an ELISA assay as described previously
(Sigurdsson et al., "Immunization with a Non-Toxic/Non-Fibrillar
Amyloid-.beta. Homologous Peptide Reduces Alzheimer's Disease
Associated Pathology in Transgenic Mice," Am J Pathol. 159:439-447
(2001) and Sigurdsson et al., "An Attenuated Immune Response is
Sufficient to Enhance Cognition in an Alzheimer's Disease Mouse
Model Immunized with Amyloid-beta Derivatives," J Neurosci.
24:6277-6282 (2004), which are hereby incorporated by reference in
their entirety), where the immunogen was coated onto Immulon.TM.
microtiter wells (Thermo Fischer Scientific, Waltham, Mass.). For
detection, goat anti-mouse IgG (Pierce, Rockford, Ill.) or
anti-mouse IgM (Sigma, St. Louis, Mo.) linked to a horseradish
peroxidase were used at 1:3000 dilution. Tetramethyl benzidine
(Pierce) was the substrate.
[0112] FIG. 1A shows the robust IgG and IgM immune response in
JNPL3 P301L tangle mice immunized with
Tau210-216[P-Thr.sub.212-Ser.sub.214] (SEQ ID NO: 2) linked to
tetanus toxin helper T-cell epitope (TT947-967) via GPSL linker.
Mice of 2-3 months of age received the first two immunizations two
weeks apart and then monthly thereafter. To assess antibody
response, the mice were bled prior to the first immunization,
periodically thereafter one week after vaccine administration, and
when the mice were killed for tissue harvesting at 8-9 months of
age. FIG. 1A shows IgG and IgM antibody response measured one week
after the 6.sup.th immunization (T3) and again at 8-9 months of
age, which was at the time of sacrifice (Tf=Tfinal). FIG. 1B shows
that a strong antibody response was generated against the tetanus
toxin epitope itself as assessed by IgG and IgM binding to an
unrelated tau epitope Tau260-264[P-Ser.sub.262] linked via GPSL to
TT947-967.
[0113] JNPL3 P301L tangle mice immunized with
Tau260-264[P-Ser.sub.262](SEQ ID NO:3) linked to tetanus toxin
helper T-cell epitope (TT947-967) via GPSL linker generated a
robust IgG response against the immunogen at shown in FIG. 2A. As
above, the mice received the first two immunizations two weeks
apart and then monthly thereafter from 2-3 months of age until 8-9
months of age. A good portion of that antibody response is
generated against the tetanus toxin epitope as assessed by IgG
binding to an unrelated tau epitope
Tau210-216[P-Thr.sub.212-Ser.sub.214] linked via GPSL to TT947-967
(FIG. 2B). However, as shown in FIG. 2C, a good portion of the
antibody response is also generated against the tau epitope as
assessed by IgG binding to a larger tau epitope
Tau240-270[P-Ser.sub.262] that contains the
Tau260-264[P-Ser.sub.262] region. T0-Tfinal: Bleed prior to
vaccination (T0), one week after third -(T1), sixth -(T2), seventh
(T3) immunization, and at tissue harvesting (Tf).
[0114] A robust antibody (IgG) response was generated in JNPL3
P301L tangle model mice immunized in with
Tau229-237[P-Thr.sub.231-Ser.sub.235] (SEQ ID NO: 4) linked to
tetanus toxin helper T-cell epitope (TT947-967] (FIG. 3). The mice
were immunized from 2-3 months of age, two weeks apart and a month
later, and bled (T1) one week after the third immunization.
[0115] A robust antibody (IgG) response was also generated in JNPL3
P301L tangle model mice immunized with the pseudophosphorylated
immunogen, Tau379-408[Asp.sub.396, 404] (SEQ ID NO: 57) in alum
adjuvant. Importantly, these antibodies recognize the
phospho-epitope, Tau379-408[P-Ser.sub.396, 404], to a similar
degree. The mice were immunized from 2-3 months of age, every two
weeks for the first two immunizations, and monthly thereafter. The
mice were bled (Tf=Tfinal) at the time of tissue harvesting at 7-8
months of age.
Example 5
Tau Immunotherapy Reduces Tau Aggregation in the Brain
[0116] For histological analysis of tau pathology, mice were
anesthetized with sodium pentobarbital (120 mg/kg, i.p.), perfused
transaortically with PBS and the brains processed as previously
described (Sigurdsson et al., "Immunization with a
Non-Toxic/Non-Fibrillar Amyloid-.beta. Homologous Peptide Reduces
Alzheimer's Disease Associated Pathology in Transgenic Mice," Am J
Pathol 159:439-447 (2001); Sigurdsson et al., "An Attenuated Immune
Response is Sufficient to Enhance Cognition in an Alzheimer's
Disease Mouse Model Immunized with Amyloid-beta Derivatives," J
Neurosci 24:6277-6282 (2004); and Sigurdsson E., "Histological
Staining of Amyloid-beta in Mouse Brains," Methods Mol Biol
299:299-308 (2005), which are hereby incorporated by reference in
their entirety). Briefly, the right hemisphere was immersion fixed
overnight in periodate-lysine-paraformaldehyde (PLP), whereas the
left hemisphere was snap-frozen for tau protein analysis. Following
fixation, the brain was moved to a phosphate buffer solution
containing 20% glycerol and 2% dimethylsulfoxide (DMSO) and stored
at 4.degree. C. until sectioned. Serial coronal brain sections (40
.mu.m) were cut and every tenth section was stained with the PHF1
monoclonal antibody that recognizes phosphorylated serines 396 and
404 located within the microtubule-binding repeat on the C-terminal
of PHF tau protein (Otvos et al., "Monoclonal Antibody PHF-1
Recognizes Tau Protein Phosphorylated at Serine Residues 396 and
404," J Neurosci Res 39:669-673 (1994), which is hereby
incorporated by reference in its entirety)
[0117] Tau antibody staining was performed as described in
Sigurdsson et al., "Immunization with a Non-Toxic/Non-Fibrillar
Amyloid-.beta. Homologous Peptide Reduces Alzheimer's Disease
Associated Pathology in Transgenic Mice," Am J Pathol 159:439-447
(2001) and Sigurdsson et al., "An Attenuated Immune Response is
Sufficient to Enhance Cognition in an Alzheimer's Disease Mouse
Model Immunized with Amyloid-beta Derivatives," J Neurosci
24:6277-6282 (2004), which are hereby incorporated by reference in
their entirety. Briefly, sections were incubated in the primary
PHF1 antibody at a 1:100 to 1:1000 dilution. A mouse on mouse
immunodetection kit (Vector Laboratories, Burlingame, Calif.) was
used, in which the anti-mouse IgG secondary antibody was used at a
1:2000 dilution.
[0118] Analysis of tissue sections was quantified with a Bioquant
image analysis system. The software uses hue, saturation, and
intensity to segment objects in the image field. Thresholds were
established with accurately identified objects on a standard set of
slides and these segmentation thresholds remained constant
throughout the analysis session. After establishing the threshold
parameter, the image field was digitized with a frame grabber. The
Bioquant software corrects for heterogeneity in background
illumination (blank field correction) and calculates the
measurement parameter for the entire field. For quantitative image
analysis of immunohistochemistry, the granular layer of the dentate
gyms was initially selected which consistently contained
intraneuronal tau aggregates (pretangles and tangles). This
observation concurs with the original characterization of this
model (Lewis et al., "Neurofibrillary Tangles, Amyotrophy and
Progressive Motor Disturbance in Mice Expressing Mutant (P301L) Tau
Protein," Nat Genet 25:402-405 (2000), which is hereby incorporated
by reference in its entirety). All procedures were performed by an
individual blind to the experimental conditions of the study.
Sample numbers were randomized before the start of the tissue
processing, and the code was broken only after the analysis was
complete. Every tenth section from the mouse brain was sampled and
the measurement was the percent of area in the measurement field at
.times.200 magnification occupied by reaction product with the tip
of the dentate gyms at the left edge of the field. Four to five
sections were analyzed per animal.
[0119] Immunization of homozygous JNPL3 tau P301L mice with
Tau260-264[P-Ser.sub.262] (SEQ ID NO: 3) linked to TT947-967 (T299)
reduced the levels of pathological tau in both the brain stem (FIG.
5A) and the dentate gyms (FIG. 5B) compared to control mice
receiving adjuvant only. Similarly, immunization of htau/PS1 mice
with the phosphorylated Tau379-408[P-Ser.sub.396,404] immunogenic
peptide reduced the amount of tau aggregates by 56% in the pyriform
cortex (FIG. 6, compare htau/PS1 immunized vs. htau/PS1 controls).
Significant difference was observed between the immunized and
control groups (one-way ANOVA, p<0.01). Post hoc analysis also
showed that immunized htau/PS1 mice differed from their htau/PS1
controls (p<0.01). ** p<0.01.
Example 6
Tau Immunotherapy Prevents Cognitive Decline
[0120] To determine if the tau immunotherapy prevented or reversed
the age-related sensorimotor abnormalities observed in the P301L or
if it caused any motor impairments in the htau/PS1 mice, animals
administered the immunogenic Tau 260-264[P-Ser.sub.262] (SEQ ID NO:
3) or Tau 379-408[P-Ser.sub.396, 404] (SEQ ID NO: 82) were assessed
using a variety of sensorimotor and cognitive tests described
below.
[0121] Rotarod Test: Animals were placed onto the rod (diameter 3.6
cm) apparatus to assess differences in motor coordination and
balance by measuring fore- and hindlimb motor coordination and
balance (Rotarod 7650 accelerating model; Ugo Basile, Biological
Research Apparatus, Varese, Italy). This procedure was designed to
assess motor behavior without a practice confound. The animals were
habituated to the apparatus by receiving training sessions of two
trials, sufficient to reach a baseline level of performance. Then,
the mice were tested three additional times, with increasing speed.
During habituation, the rotarod was set at 1.0 rpm, which was
gradually raised every 30 sec, and was also wiped clean with 30%
ethanol solution after each session. A soft foam cushion was placed
beneath the apparatus to prevent potential injury from falling.
Each animal was tested for three sessions (data combined for
subsequent analysis), with each session separated by 15 min, and
measures were taken for latency to fall or invert (by clinging)
from the top of the rotating barrel.
[0122] Traverse Beam: This task tests balance and general motor
coordination and function integration. Mice were assessed by
measuring their ability to traverse a graded narrow wooden beam to
reach a goal box (Tones et al., "Behavioural, Histochemical and
Biochemical Consequences of Selective Immunolesions in Discrete
Regions of the Basal Forebrain Cholinergic System," Neuroscience
63:95-122 (1994), which is hereby incorporated by reference in its
entirety). The mice were placed on a 1.1 cm wide beam that is 50.8
cm long and suspended 30 cm above a padded surface by two identical
columns. Attached at each end of the beam is a shaded goal box.
Mice were placed on the beam in a perpendicular orientation to
habituate and were then monitored for a maximum of 60 sec. The
number of foot slips each mouse had before falling or reaching the
goal box were recorded for each of four successive trials. Errors
are defined as footslips and were recorded numerically.
[0123] Radial Arm Maze: The maze apparatus is an 8-arm elevated
radial maze constructed from Plexiglas. Each arm is 35 cm long and
7 cm wide with a water cup 1 cm in diameter positioned at the end
of each arm. Sidewalls 15 cm high extend 12 cm into each arm to
prevent animals from crossing between arms. The central area is an
octagonal shaped hub 14 cm in diameter. Clear Plexiglas guillotine
doors, operated remotely by a pulley system control access to the
arms. The maze is elevated 75 cm above floor level and situated in
a room in which several distinctive objects of a constant location
serve as extra maze cues. Prior to testing, mice were adapted for 5
days. During this period, the mice received 0.1% saccharine in
water for 1 hour per day and were then adapted 16 hours later to
access the sugar solution from a cup placed at the end of each arm.
The first two days of adaptation were performed in a Y-maze which
the mice were allowed to explore freely. The subsequent three days
of adaptation were performed in the radial arm maze, in which the
doors were raised and lowered periodically to accustom the animals
to the sound associated with their operation. The same water
deprivation schedule was maintained during the 9 day testing
period. The mice maintain good health on this schedule. Each
testing trial was begun by placing the mouse in the central area
and raising all doors. When an arm was entered all doors were
lowered. After the mouse consumed the saccharine water, the door to
that arm was raised allowing the mouse to return to the central
arena. After a 5 sec interval, the next trial was initiated by
again raising all of the doors simultaneously. This procedure was
continued until the animal had entered all 8 arms or until 10 min
has elapsed. Daily acquisition sessions were continued for 9 days.
The number of errors (entries to previously visited arms) and time
to complete each session were recorded.
[0124] Object Recognition: The spontaneous object recognition test
that was utilized measures deficits in short term memory, and was
conducted in a square-shaped open-field box (48 cm square, with 18
cm high walls constructed from black Plexiglas), raised 50 cm from
the floor. The light intensity was set to 30 lx. On the day before
the tests, mice were individually habituated in a session in which
they were allowed to explore the empty box for 15 min. During
training sessions, two novel objects were placed at diagonal
corners in the open field and the animal was allowed to explore for
15 min. For any given trial, the objects in a pair were 10 cm high,
and composed of the same material so that they could not readily be
distinguished by olfactory cues. The time spent exploring each
object was recorded by a tracking system (San Diego Instruments,
San Diego, Calif.), and at the end of the training phase, the mouse
was removed from the box for the duration of the retention delay
(RD=3 h). Normal mice remember a specific object after a delay of
up to 1 h and spend the majority of their time investigating the
novel object during the retention trial. During retention tests,
the animals were placed back into the same box, in which one of the
previous familiar objects used during training was replaced by a
second novel object, and allowed to explore freely for 6 min. A
different object pair was used for each trial for a given animal,
and the order of exposure to object pairs as well as the designated
sample and novel objects for each pair were counterbalanced within
and across groups. The time spent exploring the novel and familiar
objects was recorded for the 6 min.
[0125] Closed Field Symmetrical Maze: This apparatus is a
rectangular field 30 cm square with 9 cm high walls divided into
36, 9.5 cm squares and covered by a clear Plexiglas top. Endboxes,
each 11.times.16.times.9 cm, are situated at diagonal corners of
the field. The symmetrical maze is a modification of the
Hebb-Williams and Rabinovitch-Rosvold type of tests, as discussed
previously (Asuni et al., "Vaccination of Alzheimer's Model Mice
with Abeta Derivative in Alum Adjuvant Reduces Abeta Burden without
Microhemorrhages," Eur J Neurosci 24:2530-2542 (2006), which is
hereby incorporated by reference in its entirety). Briefly, the
main difference is that each end-compartment functions as both a
startbox and a goalbox, and the mice run in opposite direction on
alternate trials, thereby eliminating intertrial handling. The
barriers are placed in the field in symmetrical patterns, so that
mice face the same turns going in either direction within a given
problem. Prior to testing, the mice were adapted to a water
restriction schedule (2 h daily access to water). The mice were
given two adaptation sessions prior to the beginning of testing. In
the first session, all animals were given saccharine flavored water
in the goal box for 10 min. In session 2, they were placed in the
start chamber and permitted to explore the field and enter the goal
box where water reward (0.05 mL) was available. When the mice were
running reliably from the start chamber to the goal box, they were
given three practice sessions on simple problems where one or two
barriers were placed in different positions in the field so as to
obstruct direct access to the goal box. Formal testing consisted of
the presentation of three problems graded in difficulty based on
previous data (Asuni et al., "Vaccination of Alzheimer's Model Mice
with Abeta Derivative in Alum Adjuvant Reduces Abeta Burden without
Microhemorrhages," Eur J Neurosci 24:2530-2542 (2006), which is
hereby incorporated by reference in its entirety) and published
norms for mice. One problem was presented per day and the mice were
given five trials on each problem with an intertrial interval of 2
min. Performance was scored manually by the same observer in terms
of errors (i.e., entries and reentries into designated error zones)
and time to complete each trial.
[0126] The objective of these experiments was to evaluate the
effects of the vaccination on selected sensorimotor (i.e., traverse
beam and rotarod) and cognitive behaviors (i.e., radial arm maze,
object recognition test, and closed field symmetrical maze test).
The homozygous P301L mice have tangle pathology as early as 3
months of age and those animals were tested at 5 and 8 months of
age. The htau/PS1 animals were tested at 7-8 months of age.
[0127] Immunization of homozygous JNPL3 tau P301L mice with the
phosphorylated immunogenic tau peptide Tau260-264[P-Ser.sub.262]
linked to the tetanus toxin helper T-cell epitope (TT947-967)
prevented functional impairment associated with the development of
neurofibrillary tangles as assessed using the traverse beam test at
8 months of age (FIG. 7A) and the rotarod test at 5-6 months of age
and at 8-9 months of age (FIG. 7B). Control JNPL3 tau P301L mice
received adjuvant alone.
[0128] Immunization of htau/PS1 mice with the phosphorylated
Tau379-408[P-Ser396,404] prevented cognitive decline in all three
tests that were employed: 1) the radial arm maze (RAM; two-way
ANOVA repeated measures, p<0.0001, FIG. 8A), 2) the object
recognition test (ORT; one-way ANOVA, p=0.005, FIG. 8B), and 3) the
closed field symmetrical maze (CFSM; one-way ANOVA, Maze A:
p<0.001, Maze B: p<0.0001, Maze C: p<0.01, FIGS. 9A-9C).
In the RAM and the CFSM, the immunized htau/PS1 mice performed
better than the control htau/PS1 mice on all the days (RAM;
p<0.01-0.001) and in all the mazes that were of increasing
complexity, as indicated by the number of errors (note that the Y
axis scale differs; CFSM Maze A: p<0.01, Mazes B, C:
p<0.001). In the ORT, post hoc analysis revealed that the
immunized htau/PS1 mice had better short-term memory than identical
control mice (p<0.01). It is well established that cognitively
normal mice spend about 70% of their time with the new object
compared to the old object (Asuni et al., "Immunotherapy Targeting
Pathological Tau Conformers in a Tangle Mouse Model Reduces Brain
Pathology with Associated Functional Improvements," J Neurosci
27:9115-9129 (2007), which is hereby incorporated by reference in
its entirety). The immunized htau/PS1 mice did not differ
significantly from their non-immunized identical control mice in
any of the sensorimotor tasks (rotarod, traverse beam, locomotor
activity). These findings indicate that the cognitive improvements
observed following the immunization cannot be explained by
sensorimotor effects, which further strengthens the results.
Example 7
Tau Immunotherapy Reduces Levels of Pathological Tau
[0129] Brain tissue was homogenized in a buffer containing 0.1 mM
2-(N-morpholino) ethanosulfonic acid, 0.5 mM MgSO.sub.4, 1 mM EGTA,
2 mM dithiothreitol, pH 6.8, 0.75 mM NaCl, 2 mM phenylmethyl
sulfonyl fluoride, Complete mini protease inhibitor mixture (1
tablet in 10 ml of water; Roche) and phosphatase inhibitors (20 mM
NaF and 0.5 mM sodium orthovanadate). The homogenate was then
centrifuged (20,000.times.g) for 30 min at 4.degree. C. to separate
a soluble cytosolic fraction (supernatant 1) and insoluble fraction
(pellet 1). The pellet was resuspended in the same volume of buffer
without protease and phosphatase inhibitors, but that contained 1%
(v/v) Triton X-100 and 0.25% (w/v) desoxycholate sodium and
ultracentrifuged at 50,000 for 30 min to obtain a
detergent-extracted supernatant 2 that was analyzed as insoluble
fraction. Supernatant 1 and 2 were heated at 100.degree. C. for 5
min and the same amount of protein was electrophoresed on 12% (w/v)
polyacrylamide gel. The blots were blocked in 5% non-fat milk with
0.1% Tween-20 in TBS, and incubated with different antibodies
overnight, and then washed and incubated at room temperature for 1
h with peroxidase-conjugated, anti-mouse or anti-rabbit IgG.
Subsequently, the bound antibodies were detected by ECL (Pierce).
Densitometric analysis of immunoblots were performed by NIH Image J
program and the levels of pathological tau was normalized relative
to the amounts of total tau protein instead of actin levels, as
some studies have reported that changes in pathophysiological
conditions and interactions with extracellular matrix components
can alter actin protein synthesis, rendering actin unsuitable as an
internal standard.
[0130] For Western blot analysis, total tau was measured with
polyclonal B19 antibody whereas pathological tau was detected with
monoclonal PHF 1 antibody (FIGS. 10A-10F). Levels of total soluble
and insoluble tau did not differ significantly between the groups
(FIG. 10A-10B), whereas levels of soluble PHF1 stained tau were
significantly decreased (41%, p<0.001) in the immunized mice
compared to their identical controls (FIG. 10C). A trend was
observed for a decrease (22%) in insoluble PHF1 reactive tau (FIG.
10D). Further analysis indicated a very strong trend for the
immunotherapy to reduce the ratio of PHF1/B19 by 35% and 43% in the
soluble and insoluble fractions, respectively (FIGS. 10E and 10F).
These findings indicate that pathological tau was preferentially
being cleared.
[0131] Importantly, cognitive improvements observed in the htau/PS1
mice receiving immunotherapy correlated well with reduction in PHF
1 stained tau aggregates assessed by immunohistochemistry.
Significant correlation was observed in all three memory tests (RAM
(last day of testing analyzed): r=0.36, p=0.01; CFSM: Maze A,
r=0.33, p=0.02; Maze C, r=0.40, p=0.01; ORT: r=-0.31, p=0.03). With
regard to the western blot fractions, significant correlation was
observed in both soluble and insoluble fractions and their ratios
relative to total tau in the radial arm maze (soluble PHF1: r=0.41,
p<0.01; soluble PHF1/total soluble tau: r=0.34, p<0.05;
insoluble PHF1: r=0.52, p<0.001; insoluble PHF1/total insoluble
tau: r=0.33, p<0.05) but not in the two other cognitive
tests.
Example 8
Passive Immunotherapy Targeting the P-396, 404 Epitope Prevents
Functional Decline and Reduces Tau Aggregates in the Brain
[0132] To determine the feasibility of passive immunotherapy,
homozygous P301L mice were injected intraperitoneally (i.p.) with
PHF1, a monoclonal tau antibody (provided by Dr. Peter Davies) that
recognizes NFT and pretangles in the P301L (JNPL3) mouse model and
in AD (Lewis et al, "Neurofibrillary Tangles, Amyotrophy and
Progressive Motor Disturbance in Mice Expressing Mutant (P301L) Tau
Protein," Nat Genet 25:402-40522 (2000), which is hereby
incorporated by reference in its entirety). This monoclonal
antibody recognizes tau that is phosphorylated on serine amino
acids 404 and 396 on the C-terminal of tau (Greenberg et al.,
"Hydrofluoric Acid-Treated Tau PHF Proteins Display the Same
Biochemical Properties as Normal Tau," J Biol Chem 267:564-569
(1992) which is hereby incorporated by reference in its entirety).
Therefore, it is a monoclonal analog of the prototype of one active
immunization approach (Asuni et al., "Immunotherapy Targeting
Pathological Tau Conformers in a Tangle Mouse Model Reduces Brain
Pathology with Associated Functional Improvements," J Neurosci
27:9115-9129 (2007), which is hereby incorporated by reference in
its entirety), Tau379-408[P-Ser396,404] that contains the PHF1
antibody epitope.
[0133] The dose of PHF1 was 250 .mu.g/125 .mu.L dissolved in PBS.
Controls were injected i.p. with same dose of mouse IgG in PBS. The
first injection was administered between 9-12 weeks of age. Animals
subsequently received weekly administrations for a total of 13
injections, followed by behavioral testing at 5-6 months and
subsequent tissue analysis at 6-7 months.
[0134] Passive immunization with the PHF1 antibody prevented tau
pathology associated motor decline in the P301L mouse model. As
shown in FIG. 11A, there was a significant difference between IgG
injected controls and PHF1 immunized animals on the traverse beam,
with control animals having more footslips when crossing the beam
than immunized animals (trials combined, p=0.03). Likewise, PHF1
immunized P301L mice had 58% less PHF1 stained tau pathology in the
dentate gyrus than controls (p=0.02) (FIG. 11B). An inverse
correlation between plasma levels of PHF 1 antibodies and tau
pathology was observed in the brain stem (FIG. 12A; p<0.01), and
a strong trend for correlation in the motor cortex (FIG. 12B;
p=0.06).
[0135] The amount of PHF-1 antibodies (.mu.g/.mu.L) in plasma of
immunized animals decreased four-fold in two weeks (FIG. 11C). No
detectable antibodies were observed in controls. These are the
average values for the immunized mice.
Example 9
Generation of Monoclonal Tau Antibodies
[0136] Ten balb/c mice were immunized with
Tau386-408[P-Ser.sub.396,404] (SEQ ID NO:13) linked to KLH via a
cysteine residue added to the N-terminus. Strong antibody titer was
generated against the tau portion of the immunogen as detected by
serial dilutions of plasma (FIG. 13A). Two mice were selected for
cell fusion and initial screening was performed with the immunogen
peptide without KLH. Second screening was performed with the same
peptide as well as Tau386-408[P-Ser.sub.396],
Tau386-408[P-Ser.sub.404] and the non-phospho peptide Tau386-408
(FIG. 13B). Based on that screening, clones were selected for the
first and second subcloning. Importantly, numerous strongly
positive clones were identified (>50) and stable clones have
been identified that specifically recognize a phospho-epitope
within this region or that bind to a non-phosphorylated site within
this region, thereby allowing a comparison of the efficacy and
safety profile of antibodies binding to a phospho- or non-phospho
tau epitopes within the same region of the molecule.
[0137] Of the phospho-specific monoclonal antibodies selected for
further subcloning, four out of six retained their specificity for
the phospho-Ser.sub.404 epitope (see clones 1F12C2, 1F12G6, 4E6E3,
and 4E6G7 in FIG. 14A). Two clones are less phospho-specific
(8B2D1) or non-specific (8B2D4) (FIG. 14A). Of the
non-phospho-specific monoclonal antibodies, 6B2E9 and 6B2G12, in
particular, retained their non-specificity after further subcloning
(FIG. 14B).
[0138] The reactivity of the four P-Ser.sub.396, 404 tau
phospho-specific (FIG. 15A) and non-phospho-specific (FIG. 15B)
monoclonal antibody clones was tested against brain homogenates
from the JNPL3 P301L mouse and wildtype (Wt) mouse. Of the four
phospho-specific clones, 4E6G7 shows the strongest reactivity (FIG.
15A), which is consistent with the ELISA results of FIG. 14A. In
contrast with the PHF-1 antibody that also recognizes the tau
P-Ser.sub.396, 404 epitope, all clones react better with the JNPL3
P301L brain homogenate than the Wt homogenate. The
non-phospho-specific clones reacted faster, as expected, as most of
tau is non-phosphorylated.
[0139] Another set of ten balb/c mice was immunized with
Tau260-271[P-Ser.sub.262](SEQ ID NO:12) linked to KLH via a
cysteine residue on the C-terminus. Although strong titer was
generated against the Tau260-271[P-Ser.sub.262] immunogen, plasma
antibodies recognized the non-phospho peptide Tau260-271 as well
(FIG. 16A). Eight stable phospho-specific clones were selected from
the second subcloning for further analysis (FIG. 16B) and the 2C11
clone has been selected for antibody production as it is of the
IgG2a isotype. IgG3 has shorter half-life and is therefore not
considered ideal for passive immunization studies.
[0140] The reactivity of the three phospho-specific P-Ser.sub.262
tau monoclonal antibody clones against brain homogenates from JNPL3
P301L and wildtype (Wt) mice was assessed (FIG. 17). The 2C11
antibody clone recognizes a higher molecular weight band than the
other phospho-specific clones and it does not distinguish between
wildtype and P301L tissue. 5F7D10 and 5F7E9 are representatives of
the other clones. Tau-5 recognizes total tau and binds to an
epitope around amino acids 216-227 of tau. CP27 recognizes human
but not mouse tau.
[0141] The 5F7D10 antibody clone readily detected tau pathology in
P301L tangle mouse brain sections as shown in FIGS. 18A-18E. The
5F7D10 monoclonal antibody shows strong histological staining in
the P301L brain section (FIG. 18A) compared to the wildtype (FIG.
18B). The PHF1 antibody picked up tau pathology in the same tangle
mouse (FIG. 18C) although the pattern was different than with the
5F7D10 antibody, which is not surprising as they recognize
different tau epitopes. FIG. 18D is a magnified image of the boxed
region in FIG. 18A depicting neurons with aggregated tau. FIG. 18E
is a higher magnified image of tangle-like pathology detected with
5F7D10 in a different JNPL3 P301L mouse.
[0142] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these are therefore considered to be
within the scope of the invention as defined in the claims which
follow.
Sequence CWU 1
1
1031441PRTHomo Sapiens 1Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met
Glu Asp His Ala Gly1 5 10 15Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln
Gly Gly Tyr Thr Met His 20 25 30Gln Asp Gln Glu Gly Asp Thr Asp Ala
Gly Leu Lys Glu Ser Pro Leu 35 40 45Gln Thr Pro Thr Glu Asp Gly Ser
Glu Glu Pro Gly Ser Glu Thr Ser 50 55 60Asp Ala Lys Ser Thr Pro Thr
Ala Glu Asp Val Thr Ala Pro Leu Val65 70 75 80Asp Glu Gly Ala Pro
Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu 85 90 95Ile Pro Glu Gly
Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro 100 105 110Ser Leu
Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val 115 120
125Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly
130 135 140Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala
Pro Pro145 150 155 160Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile
Pro Ala Lys Thr Pro 165 170 175Pro Ala Pro Lys Thr Pro Pro Ser Ser
Gly Glu Pro Pro Lys Ser Gly 180 185 190Asp Arg Ser Gly Tyr Ser Ser
Pro Gly Ser Pro Gly Thr Pro Gly Ser 195 200 205Arg Ser Arg Thr Pro
Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys 210 215 220Lys Val Ala
Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys225 230 235
240Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val
245 250 255Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro
Gly Gly 260 265 270Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu
Ser Asn Val Gln 275 280 285Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys
His Val Pro Gly Gly Gly 290 295 300Ser Val Gln Ile Val Tyr Lys Pro
Val Asp Leu Ser Lys Val Thr Ser305 310 315 320Lys Cys Gly Ser Leu
Gly Asn Ile His His Lys Pro Gly Gly Gly Gln 325 330 335Val Glu Val
Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser 340 345 350Lys
Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn 355 360
365Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala
370 375 380Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val
Val Ser385 390 395 400Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val
Ser Ser Thr Gly Ser 405 410 415Ile Asp Met Val Asp Ser Pro Gln Leu
Ala Thr Leu Ala Asp Glu Val 420 425 430Ser Ala Ser Leu Ala Lys Gln
Gly Leu 435 44027PRTArtificialtau peptide 2Ser Arg Thr Pro Ser Leu
Pro1 535PRTArtificialtau peptide 3Ile Gly Ser Thr Glu1
549PRTArtificialtau peptide 4Val Arg Thr Pro Pro Lys Ser Pro Ser1
5513PRTArtificialtau peptide 5Tyr Lys Ser Pro Val Val Ser Gly Asp
Thr Ser Pro Arg1 5 10630PRTArtificialtau peptide 6Gly Asp Arg Ser
Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly1 5 10 15Ser Arg Ser
Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg 20 25
30730PRTArtificialtau peptide 7Gly Asp Arg Ser Gly Tyr Ser Ser Pro
Gly Ser Pro Gly Thr Pro Gly1 5 10 15Ser Arg Ser Arg Thr Pro Ser Leu
Pro Thr Pro Pro Thr Arg 20 25 30830PRTArtificialtau peptide 8Gly
Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly1 5 10
15Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg 20 25
30930PRTArtificialtau peptide 9Gly Asp Arg Ser Gly Tyr Ser Ser Pro
Gly Ser Pro Gly Thr Pro Gly1 5 10 15Ser Arg Ser Arg Thr Pro Ser Leu
Pro Thr Pro Pro Thr Arg 20 25 301030PRTArtificialtau peptide 10Pro
Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu1 5 10
15Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val 20 25
301137PRTArtificialtau peptide 11Cys Gly Ser Leu Gly Asn Ile His
His Lys Pro Gly Gly Gly Gln Val1 5 10 15Glu Val Lys Ser Glu Lys Leu
Asp Phe Lys Asp Arg Val Gln Ser Lys 20 25 30Ile Gly Ser Leu Asp
351212PRTArtificialtau peptide 12Ile Gly Ser Thr Glu Asn Leu Lys
His Gln Pro Gly1 5 101323PRTArtificialtau peptide 13Thr Asp His Gly
Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly1 5 10 15Asp Thr Ser
Pro Arg His Leu 201424PRTArtificialtau peptide 14Leu Gln Thr Pro
Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr1 5 10 15Ser Asp Ala
Lys Ser Thr Pro Thr 20155PRTArtificialtau peptide 15Thr Pro Ser Leu
Glu1 5165PRTArtificialtau peptide 16Ile Ala Thr Pro Arg1
5175PRTArtificialtau peptide 17Ala Lys Thr Pro Pro1
51817PRTArtificialtau peptide 18Pro Gly Thr Pro Gly Ser Arg Ser Arg
Thr Pro Ser Leu Pro Thr Pro1 5 10 15Pro195PRTArtificialtau peptide
19Pro Lys Ser Pro Ser1 5209PRTArtificialtau peptide 20Val Lys Ser
Lys Ile Gly Ser Thr Glu1 5215PRTArtificialtau peptide 21Val Gln Ser
Lys Cys1 5225PRTArtificialtau peptide 22Ile Gly Ser Leu Asp1
52319PRTArtificialtau peptide 23Val Val Ser Gly Asp Thr Ser Pro Arg
His Leu Ser Asn Val Ser Ser1 5 10 15Thr Gly Ser2418PRTArtificialtau
peptide 24Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val Ser
Ala Ser1 5 10 15Leu Ala254PRTArtificialtau peptide 25Pro Gly Ser
Pro1265PRTArtificialtau peptide 26Pro Gly Thr Pro Gly1
52711PRTArtificialtau peptide 27Tyr Ser Ser Pro Gly Ser Pro Gly Thr
Pro Gly1 5 102811PRTArtificialtau peptide 28Pro Gly Ser Arg Ser Arg
Thr Pro Ser Leu Pro1 5 102911PRTArtificialtau peptide 29Val Arg Thr
Pro Pro Lys Ser Pro Ser Ser Ala1 5 103010PRTArtificialtau peptide
30Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro1 5
10317PRTArtificialpseudophosphorylated tau peptide 31Ser Arg Xaa
Pro Xaa Leu Pro1 5325PRTArtificialpseudophosphorylated tau peptide
32Ile Gly Xaa Thr Glu1 5339PRTArtificialpseudophosphorylated tau
peptide 33Val Arg Xaa Pro Pro Lys Xaa Pro Ser1
53413PRTArtificialpseudophosphorylated tau peptide 34Tyr Lys Xaa
Pro Val Val Ser Gly Asp Thr Xaa Pro Arg1 5
103530PRTArtificialpseudophosphorylated tau peptide 35Gly Asp Arg
Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly1 5 10 15Ser Arg
Ser Arg Xaa Pro Xaa Leu Pro Thr Pro Pro Thr Arg 20 25
303630PRTArtificialpseudophosphorylated tau peptide 36Gly Asp Arg
Ser Gly Tyr Ser Xaa Pro Gly Xaa Pro Gly Xaa Pro Gly1 5 10 15Ser Arg
Ser Arg Xaa Pro Xaa Leu Pro Thr Pro Pro Thr Arg 20 25
303730PRTArtificialpseudophosphorylated tau peptide 37Gly Asp Arg
Ser Gly Tyr Ser Xaa Pro Gly Ser Pro Gly Thr Pro Gly1 5 10 15Ser Arg
Ser Arg Xaa Pro Xaa Leu Pro Xaa Pro Pro Thr Arg 20 25
303830PRTArtificialpseudophosphorylated tau peptide 38Gly Asp Arg
Ser Gly Tyr Ser Ser Pro Gly Xaa Pro Gly Xaa Pro Gly1 5 10 15Ser Arg
Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg 20 25
303930PRTArtificialpseudophosphorylated tau peptide 39Pro Gly Ser
Pro Gly Thr Pro Gly Ser Arg Ser Arg Xaa Pro Xaa Leu1 5 10 15Pro Thr
Pro Pro Thr Arg Glu Pro Lys Lys Val Ala Val Val 20 25
304037PRTArtificialpseudophosphorylated tau peptide 40Cys Gly Xaa
Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln Val1 5 10 15Glu Val
Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys 20 25 30Ile
Gly Xaa Leu Asp 354112PRTArtificialpseudophosphorylated tau peptide
41Ile Gly Xaa Thr Glu Asn Leu Lys His Gln Pro Gly1 5
104223PRTArtificialpseudophosphorylated tau peptide 42Thr Asp His
Gly Ala Glu Ile Val Tyr Lys Xaa Pro Val Val Ser Gly1 5 10 15Asp Thr
Xaa Pro Arg His Leu 204324PRTArtificialpseudophosphorylated tau
peptide 43Leu Gln Xaa Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser
Glu Thr1 5 10 15Ser Asp Ala Lys Ser Xaa Pro Thr
20445PRTArtificialpseudophosphorylated tau peptide 44Thr Pro Xaa
Leu Glu1 5455PRTArtificialpseudophosphorylated tau peptide 45Ile
Ala Xaa Pro Arg1 5465PRTArtificialpseudophosphorylated tau peptide
46Ala Lys Xaa Pro Pro1 54717PRTArtificialpseudophosphorylated tau
peptide 47Pro Gly Xaa Pro Gly Xaa Arg Xaa Arg Xaa Pro Xaa Leu Pro
Xaa Pro1 5 10 15Pro485PRTArtificialpseudophosphorylated tau peptide
48Pro Lys Xaa Pro Ser1 5499PRTArtificialpseudophosphorylated tau
peptide 49Val Lys Xaa Lys Ile Gly Xaa Thr Glu1
5505PRTArtificialpseudophosphorylated tau peptide 50Val Gln Xaa Lys
Cys1 5515PRTArtificialpseudophosphorylated tau peptide 51Ile Gly
Xaa Leu Asp1 55219PRTArtificialpseudophosphorylated tau peptide
52Val Val Xaa Gly Asp Xaa Ser Pro Arg His Leu Xaa Asn Val Xaa Xaa1
5 10 15Xaa Gly Ser5318PRTArtificialpseudophosphorylated tau peptide
53Val Asp Xaa Pro Gln Leu Ala Xaa Leu Ala Asp Glu Val Xaa Ala Xaa1
5 10 15Leu Ala544PRTArtificialpseudophosphorylated tau peptide
54Pro Gly Xaa Pro1555PRTArtificialpseudophosphorylated tau peptide
55Pro Gly Xaa Pro Gly1 55631PRTArtificialpseudophosphorylated tau
peptide 56Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly Ala Asp
Gly Lys1 5 10 15Xaa Lys Ile Ala Xaa Thr Pro Arg Gly Ala Ala Pro Pro
Gly Gln 20 25 305730PRTArtificialpseudophosphorylated tau peptide
57Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr1
5 10 15Lys Xaa Pro Val Val Ser Gly Asp Thr Xaa Pro Arg His Leu 20
25 305830PRTArtificialpseudophosphorylated tau peptide 58Gly Asp
Arg Ser Gly Tyr Ser Xaa Pro Gly Xaa Pro Gly Xaa Pro Gly1 5 10 15Ser
Arg Ser Arg Xaa Pro Xaa Leu Pro Thr Pro Pro Thr Arg 20 25
305930PRTArtificialpseudophosphorylated tau peptide 59Arg Glu Pro
Lys Lys Val Ala Val Val Arg Xaa Pro Pro Lys Xaa Pro1 5 10 15Ser Ser
Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met 20 25
306029PRTArtificialpseudophosphorylated tau peptide 60Xaa Ser Gly
Glu Pro Pro Lys Xaa Gly Asp Arg Ser Gln Xaa Xaa Xaa1 5 10 15Pro Gly
Xaa Pro Gly Xaa Pro Gly Xaa Arg Xaa Arg Xaa 20
256130PRTArtificialpseudophosphorylated tau peptide 61Met Ala Glu
Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly1 5 10 15Thr Xaa
Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Xaa Thr 20 25
306231PRTArtificialpseudophosphorylated tau peptide 62Thr Met His
Gln Asp Gln Glu Gly Asp Xaa Asp Ala Gly Leu Lys Glu1 5 10 15Xaa Pro
Leu Gln Xaa Pro Xaa Glu Asp Gly Xaa Glu Glu Pro Gly 20 25
306331PRTArtificialpseudophosphorylated tau peptide 63Gly Ser Glu
Thr Ser Asp Ala Lys Xaa Xaa Pro Xaa Ala Glu Asp Val1 5 10 15Thr Ala
Pro Leu Val Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala 20 25
306431PRTArtificialpseudophosphorylated tau peptide 64Ala Ala Gln
Pro His Xaa Glu Ile Pro Glu Gly Xaa Xaa Ala Glu Glu1 5 10 15Ala Gly
Ile Gly Asp Thr Pro Xaa Leu Glu Asp Glu Ala Ala Gly 20 25
306531PRTArtificialpseudophosphorylated tau peptide 65Gly His Val
Xaa Gln Ala Arg Met Val Ser Lys Xaa Lys Asp Gly Thr1 5 10 15Gly Ser
Asp Asp Lys Lys Ala Lys Gly Ala Asp Gly Lys Xaa Lys 20 25
306631PRTArtificialpseudophosphorylated tau peptide 66Lys Ile Ala
Thr Pro Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln1 5 10 15Ala Asn
Ala Thr Arg Ile Pro Ala Lys Xaa Pro Pro Ala Pro Lys 20 25
306731PRTArtificialpseudophosphorylated tau peptide 67Lys Xaa Pro
Pro Xaa Xaa Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser1 5 10 15Gly Xaa
Xaa Xaa Pro Gly Xaa Pro Gly Xaa Pro Gly Xaa Arg Ser 20 25
306831PRTArtificialpseudophosphorylated tau peptide 68Ser Arg Xaa
Pro Xaa Leu Pro Xaa Pro Pro Thr Arg Glu Pro Lys Lys1 5 10 15Val Ala
Val Val Arg Xaa Pro Pro Lys Xaa Pro Xaa Xaa Ala Lys 20 25
306931PRTArtificialpseudophosphorylated tau peptide 69Lys Ser Arg
Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn1 5 10 15Val Lys
Ser Lys Ile Gly Xaa Thr Glu Asn Leu Lys His Gln Pro 20 25
307031PRTArtificialpseudophosphorylated tau peptide 70Pro Gly Gly
Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser1 5 10 15Asn Val
Gln Ser Lys Cys Gly Xaa Lys Asp Asn Ile Lys His Val 20 25
307131PRTArtificialpseudophosphorylated tau peptide 71Val Pro Gly
Gly Gly Ser Val Gln Ile Val Xaa Lys Pro Val Asp Leu1 5 10 15Ser Lys
Val Thr Ser Lys Cys Gly Xaa Leu Gly Asn Ile His His 20 25
307231PRTArtificialpseudophosphorylated tau peptide 72His Lys Pro
Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp1 5 10 15Phe Lys
Asp Arg Val Gln Ser Lys Ile Gly Xaa Leu Asp Asn Ile 20 25
307331PRTArtificialpseudophosphorylated tau peptide 73Ile Xaa His
Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Xaa His Lys1 5 10 15Leu Thr
Phe Arg Glu Asn Ala Lys Ala Lys Xaa Asp His Gly Ala 20 25
307431PRTArtificialpseudophosphorylated tau peptide 74Ala Glu Ile
Val Xaa Lys Xaa Pro Val Val Xaa Gly Asp Xaa Xaa Pro1 5 10 15Arg His
Leu Xaa Asn Val Xaa Xaa Thr Gly Ser Ile Asp Met Val 20 25
307531PRTArtificialpseudophosphorylated tau peptide 75Val Xaa Xaa
Thr Gly Ser Ile Asp Met Val Asp Xaa Pro Gln Leu Ala1 5 10 15Thr Leu
Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu 20 25
307621PRTArtificialT-helper cell epitope 76Phe Asn Asn Phe Thr Val
Ser Phe Trp Leu Arg Val Pro Lys Val Ser1 5 10 15Ala Ser His Leu Glu
207713PRTArtificialT-helper cell epitope 77Gln Tyr Ile Lys Ala Asn
Ser Lys Phe Ile Gly Ile Thr1 5 10784PRTArtificiallinker 78Gly Pro
Ser Leu1794PRTArtificiallinker 79Gly Ser Gly
Ser1805PRTArtificiallinker 80Gly Ser Gly Ser Gly1
58130PRTArtificialtau peptide 81Asp Gly Thr Gly Ser Asp Asp Lys Lys
Ala Lys Gly Ala Asp Gly Lys1 5 10 15Thr Lys Ile Ala Thr Pro Arg Gly
Ala Ala Pro Pro Gly Gln 20 25 308230PRTArtificialtau peptide 82Arg
Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr1 5 10
15Lys Ser Pro Val Val Ser
Gly Asp Thr Ser Pro Arg His Leu 20 25 308330PRTArtificialtau
peptide 83Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr
Pro Gly1 5 10 15Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr
Arg 20 25 308430PRTArtificialtau peptide 84Arg Glu Pro Lys Lys Val
Ala Val Val Arg Thr Pro Pro Lys Ser Pro1 5 10 15Ser Ser Ala Lys Ser
Arg Leu Gln Thr Ala Pro Val Pro Met 20 25 308529PRTArtificialtau
peptide 85Ser Ser Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser Gln Tyr
Ser Ser1 5 10 15Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr
20 258630PRTArtificialtau peptide 86Met Ala Glu Pro Arg Gln Glu Phe
Glu Val Met Glu Asp His Ala Gly1 5 10 15Thr Tyr Gly Leu Gly Asp Arg
Lys Asp Gln Gly Gly Tyr Thr 20 25 308731PRTArtificialtau peptide
87Thr Met His Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu1
5 10 15Ser Pro Leu Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly
20 25 308831PRTArtificialtau peptide 88Gly Ser Glu Thr Ser Asp Ala
Lys Ser Thr Pro Thr Ala Glu Asp Val1 5 10 15Thr Ala Pro Leu Val Asp
Glu Gly Ala Pro Gly Lys Gln Ala Ala 20 25 308931PRTArtificialtau
peptide 89Ala Ala Gln Pro His Thr Glu Ile Pro Glu Gly Thr Thr Ala
Glu Glu1 5 10 15Ala Gly Ile Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala
Ala Gly 20 25 309031PRTArtificialtau peptide 90Gly His Val Thr Gln
Ala Arg Met Val Ser Lys Ser Lys Asp Gly Thr1 5 10 15Gly Ser Asp Asp
Lys Lys Ala Lys Gly Ala Asp Gly Lys Thr Lys 20 25
309131PRTArtificialtau peptide 91Lys Ile Ala Thr Pro Arg Gly Ala
Ala Pro Pro Gly Gln Lys Gly Gln1 5 10 15Ala Asn Ala Thr Arg Ile Pro
Ala Lys Thr Pro Pro Ala Pro Lys 20 25 309231PRTArtificialtau
peptide 92Lys Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly Asp
Arg Ser1 5 10 15Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser
Arg Ser 20 25 309331PRTArtificialtau peptide 93Ser Arg Thr Pro Ser
Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys1 5 10 15Val Ala Val Val
Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys 20 25
309431PRTArtificialtau peptide 94Lys Ser Arg Leu Gln Thr Ala Pro
Val Pro Met Pro Asp Leu Lys Asn1 5 10 15Val Lys Ser Lys Ile Gly Ser
Thr Glu Asn Leu Lys His Gln Pro 20 25 309531PRTArtificialtau
peptide 95Pro Gly Gly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp
Leu Ser1 5 10 15Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys
His Val 20 25 309631PRTArtificialtau peptide 96Val Pro Gly Gly Gly
Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu1 5 10 15Ser Lys Val Thr
Ser Lys Cys Gly Ser Leu Gly Asn Ile His His 20 25
309731PRTArtificialtau peptide 97His Lys Pro Gly Gly Gly Gln Val
Glu Val Lys Ser Glu Lys Leu Asp1 5 10 15Phe Lys Asp Arg Val Gln Ser
Lys Ile Gly Ser Leu Asp Asn Ile 20 25 309831PRTArtificialtau
peptide 98Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys Ile Glu Thr
His Lys1 5 10 15Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr Asp His
Gly Ala 20 25 309931PRTArtificialtau peptide 99Ala Glu Ile Val Tyr
Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro1 5 10 15Arg His Leu Ser
Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val 20 25
3010031PRTArtificialtau peptide 100Val Ser Ser Thr Gly Ser Ile Asp
Met Val Asp Ser Pro Gln Leu Ala1 5 10 15Thr Leu Ala Asp Glu Val Ser
Ala Ser Leu Ala Lys Gln Gly Leu 20 25 3010115PRTArtificialtau
peptide 101His Leu Ser Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val
Asp1 5 10 1510213PRTArtificialtau peptide 102Arg Glu Asn Ala Lys
Ala Lys Thr Asp His Gly Ala Glu1 5 10103186PRTArtificialtau peptide
103Glu Ser Pro Leu Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly1
5 10 15Ser Glu Thr Ser Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val
Thr 20 25 30Ala Pro Leu Val Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala
Ala Gln 35 40 45Pro His Thr Glu Ile Pro Glu Gly Thr Thr Ala Glu Glu
Ala Gly Ile 50 55 60Gly Asp Thr Pro Ser Leu Glu Asp Glu Ala Ala Gly
His Val Thr Gln65 70 75 80Ala Arg Met Val Ser Lys Ser Lys Asp Gly
Thr Gly Ser Asp Asp Lys 85 90 95Lys Ala Lys Gly Ala Asp Gly Lys Thr
Lys Ile Ala Thr Pro Arg Gly 100 105 110Ala Ala Pro Pro Gly Gln Lys
Gly Gln Ala Asn Ala Thr Arg Ile Pro 115 120 125Ala Lys Thr Pro Pro
Ala Pro Lys Thr Pro Pro Ser Ser Gly Glu Pro 130 135 140Pro Lys Ser
Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly145 150 155
160Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr
165 170 175Arg Glu Pro Lys Lys Val Ala Val Val Arg 180 185
* * * * *